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65b890af66c13817298d53c6 | 0 | Ketenes comprise a unique class of cumulene molecules with the general formula RR'C=C=O. Initially described by Wedekind as a reactive intermediate in 1901, Staudinger reported the first synthesis and characterization of a ketene molecule, diphenyl ketene (R=R'=Ph), in 1905. Since that time, despite their relative instability, ketenes have served as valuable reagents and intermediates in organic synthesis. Currently, however, the smallest homolog (R=R'=H, ketene, ethenone) of the series is receiving attention as a potentially significant public health hazard, since it has been identified in aerosols generated by commercial vaping products. The chemical and toxicological properties of ketene mirror those of phosgene (Cl 2 C=O), a WW-I chemical warfare agent, as a reactive acylating agent and respiratory poison. There is a lack of human toxicity data for ketene exposure. The available animal data was obtained mainly prior to 1950. Exposure to animals causes alveolar damage and a delayed onset of pulmonary toxicity leading to death by pulmonary edema. Similar to phosgene, the delayed effects result from the non-enzymatic acylation of lung proteins, as opposed to direct irritation. The Acute Exposure Guideline Levels (AEGL)-3 (life-threatening levels) for ketene are 0.24 ppm for 10 min and 0.088 ppm for 8 h. Vitamin E acetate (VEA) has been linked to the 2019-2020 e-cigarette or vaping product use-associated lung injury (EVALI) epidemic, in large part due to VEA's prevalence in patient samples. In 2020, Wu and O'Shea reported the formation of ketene when heating and vaping VEA, indicating an additional possible link between ketene and EVALI. There is general agreement that EVALI is caused by chemical toxicant inhalation. However, to date, neither ketene, VEA, nor any other specific chemical has been conclusively proven to be the causative agent of EVALI. Moreover, although there has been a significant decline since 2020, cases continue to be observed throughout the US. Regardless of whether VEA-derived ketene is a primary cause of EVALI, any source of exposure to ketene may put one at risk for significant lung injury. Recently, we reported that four acetylated cannabinoids (acetylated β 8 -and β 9 -THC, CBN, and CBD; Figure ), produce ketene emissions under real-world vaping conditions, from either a vape pen or dab platform, including at levels in range of NIOSH (National Institute for Occupational Safety & Health) thresholds. Interestingly, aerosolized THC products had been reported to cause acute respiratory syndromes prior to the EVALI outbreak. 11 The strong experimental evidence for ketene emissions arising from vaping cannabinoid acetates or VEA raises two issues. First, since ketene is too reactive and shortlived to be characterized as an intact molecule under common laboratory conditions, vaping studies to date have employed the well-known method of trapping ketene with a nucleophile (i.e., benzylamine) for characterization as the corresponding N-benzylacetamide. Since amines are relatively non-selective reagents, their use for ketene trapping and determination should ideally be limited to relatively well-defined systems that do not contain molecules that can react to form the same N-benzylacetamide product as ketene. However, manufacturers are not required to disclose most vaping product ingredients and, moreover, heating and vaping produces aerosols containing complex chemical mixtures. |
65b890af66c13817298d53c6 | 1 | would result if a ketene intermediate is formed from the trideuterated acetate methyl (i.e., CBN-OAc-D 3 ). (Bottom) Conversely, if ketene is not formed as a reaction intermediate, an alternative addition-elimination or related reaction would result in trideuterated Nbenzylacetamide (N-benzylacetamide-D 3 ). The second issue centers on concerns that real-world vaping power settings do not provide enough energy to produce ketene. Recent theoretical studies show that unrealistically high vaping temperatures, in excess of at least 700 β°C, are required for any significant levels of ketene to form from VEA or other acetates. To address this issue, some have proposed that ineffective wicking (resulting in dry, overheated vaporizer coils, colloquially termed "dry puff") or catalysis are potential causes of ketene production at relatively low power settings. Alternatively, herein we hypothesize that ketene formation at real world vaping temperatures can be explained if one accounts for the impact of oxygen. This hypothesis is based on prior studies reported by our group in 2017, as well as in subsequent reports by us and by others. |
65b890af66c13817298d53c6 | 2 | Since a large portion of the volatile thermal reaction products of β 8 and β 9 THC formed under vaping conditions derive from their cyclohexene ring, we focused current experiments on CBN-acetate. CBN-acetate contains a second aryl, rather than a cyclohexene ring (Figure , see CBN-OAc-D 3 ) that renders it relatively stable, thereby reducing the analytical complexity of its corresponding aerosol samples. In addition, CBN and CBN-OAc are crystalline and thus easier to purify and handle compared to THC or CBD and their acetate derivatives. |
65b890af66c13817298d53c6 | 3 | CBN-OAc-D 3 was obtained in 85 % yield by heating CBN and acetic anhydride-D 6 under neat conditions at reflux for 2 h. Previously, Nishida et al. synthesized trideuterated phenyl acetate to obtain evidence for the thermolytic mechanism of ketene formation. They determined that a four membered ring transition state involving transfer of a hydrogen atom from the acetate methyl to the phenolic ester oxygen led to ketene. Consistent with this mechanism, we tentatively assigned the CBN-OD byproduct from deuterated ketene production shown in Scheme 1 and Figure with a deuterated phenolic oxygen. Two other prominent peaks appear at 238 amu, arising from the loss of a four-carbon subunit from the cannabinoid pentyl side chain, at 46 amu, corresponding to the trideuterated acylium ion. The appearance of the latter peak potentially serves as evidence for an alternative intermolecular pathway to ketene, for example, involving dedeuteration of the acylium ion. |
65b890af66c13817298d53c6 | 4 | In this and prior studies we used a dabbing platform to heat and aerosolize cannabinoids. Dabbing is a type of cannabis vaping that entails flash vaporizing manufactured cannabis products (e.g, high THC-or CBD-potency extracts) on a hot surface (colloquially referred to as a nail), with users often inhaling close to an entire lung volume in one puff. A national web survey of cannabis consumers found that 60% of respondents dabbed at least once, and that 38% endorsed its regular use. Apart from embodying a popular means of cannabis vaping, using a dab platform has experimental advantages compared to vape pens. For example, the aerosols produced are not as prone to the influences of solvent, viscosity, or wicking that can significantly impact vaping chemistry and aerosolization efficiency. The specific aerosolization and collection method used herein has been described by us previously, with minor changes (see Supporting Information). Briefly, the cannabinoid sample (~100 mg) is flash vaporized on a quartz dab platform surface and the generated aerosol pulled via a smoking machine into an in-line impinger. The impinger contains NMR solvent (CDCl 3 ) and the trapping agent (benzylamine) at room temperature. Aliquots of the impinger solution containing the dissolved aerosol contents generated by a single puff are directly analyzed by NMR and GC-MS. The yield of ketene (as N-benzylamide equivalents) formed from 40 mg CBN-OAc under these conditions is 0.078 Β± 0.016 mg (n=4) at 378 Β°C. 7 |
65b890af66c13817298d53c6 | 5 | The production of N-benzylacetamide-D 2 , the "trapped" ketene equivalent product shown in Scheme 1, was unambiguously confirmed. The observed ESI-HRMS peak at 150.0883 amu is within 0.7 ppm of the calculated value (150.0882 amu). Significantly, the proton-decoupled C NMR spectrum shows a characteristic quintet splitting pattern centered at 20.8 ppm for -CD 2 H, J = 20 Hz, (Figure ). |
65b890af66c13817298d53c6 | 6 | In contrast, in the vaping experiments, the 1 H NMR spectrum of the impinger solution showed the presence of N-benzylacetamide within minutes of aerosol generation and collection. Moreover, had CBN-OAc-D 3 (rather than ketene-D 2 ) directly reacted with benzylamine, a septet arising from -CD 3 splitting (from N-benzylacetamide-D 3 ) would have been observed in the C NMR, rather than the quintet shown in Figure . |
65b890af66c13817298d53c6 | 7 | Acetic anhydride reacts rapidly and irreversibly with alcohols like ethanol, especially at elevated temperatures; upon solvation any residual acetic anhydride-D 6 forms trideuterated acetic acid (AcOH-D 3 ) and trideuterated ethyl acetate (EtOAc-D 3 ), which both join the mother liquor and wash cleanly away from the CBN-OAc crystals. However, if any residual acetic anhydride-D 6 remained in the sample and was responsible for the formation of Nbenzylacetamide, the C resonance from the resultant trideuterated acetate methyl carbon would split into a septet, rather than the observed quintet. |
65b890af66c13817298d53c6 | 8 | A second control study was performed to validate the feasibility of relatively lower vaping temperatures leading to the formation of ketene. Each of the dab platform experiments described above was initially run at 378 Β°C, in keeping with our prior studies. During the course of this study, we collaborated on a peer-reviewed systematic survey and analysis of user-preferred THC-acetate dabbing temperatures. Temperatures β₯ 378 Β°C were preferred by 8 % of respondents. For relevance to a wider range of users we thus investigated and found readily detectable ( 1 H NMR, Figure ) levels of ketene as N-benzylacetamide equivalents when the aerosolization was performed at 287 Β°C, a temperature setting at or above which > To investigate the potential role of oxygen in ketene formation, we dabbed ethyl acetate (EtOAc, Figure ) and geranyl acetate (Figure ) at 378 Β°C in a glove bag in ambient air (~21% O 2 ) conditions and also under N 2 (<0.1% O 2 ). EtOAc or geranyl acetate (500 ul) were used in these experiments since they are liquids, and thus more amenable to glove bag conditions. The transformation of EtOAc to ketene had been simulated by others, with vaping temperatures > 840 β°C needed to afford enough ketene to be introduced into a user's lungs. However, Figure shows that, at 378 β°C, N-benzylacetamide derived from dabbing EtOAc is ~ 10-fold higher under ambient aerobic (0.025 mg) versus reduced O 2 conditions (0.0025 mg). Importantly, EtOAc is a prevalent flavorant molecule used in tobacco e-cigarettes. It was observed to be the fifth most frequently occurring flavor chemical in a study of 277 e-liquid products. Geranyl acetate (Figure ) exhibited analogous behavior, with ~ 20 fold more ketene generated under ambient aerobic conditions. |
65b890af66c13817298d53c6 | 9 | The safety and regulation of vaping products are controversial issues, arguably among the most polarizing in the history of tobacco control. Evaluation of tobacco as well as cannabis vaping products must include an understanding of their emissions, since significant chemical reactions occur upon both product storage as well as upon heating and vaping. Determining not only the identities of toxicant emissions, but also their origins, can support efforts towards improved product safety. |
65b890af66c13817298d53c6 | 10 | Ketene is one of the most toxic vaping emissions identified to date. To address exposure it is necessary to identify ketene at its source; otherwise its high reactivity renders its determination impractical in vivo or in vitro. The use of an isotopically labeled acetate precursor (Scheme 1) enables rigorous ketene determination via C NMR splitting patterns and high resolution mass spectrometry (Figure ). |
65b890af66c13817298d53c6 | 11 | Ketene was detectable upon vaping CBN-acetate at vaping temperatures as low, to date, as 278 β°C using a dab platform (Figure ). This is consistent with our prior work, wherein ketene was detectable using a pre-filled commercial vape cartridge and a low battery power setting. However, according to theoretical simulations, ketene formation should not occur from vaping VEA, 12 cannabinoid 5 or other acetates at real-world vaping temperature settings. |
65b890af66c13817298d53c6 | 12 | Importantly, ketene is not the only aerosol toxicant whose formation under realistic vaping power settings has been questioned. For example, findings showing that e-cigarette flavorant additives were linked to elevated toxic aldehyde emissions were initially criticized by industry supporters as faulty. This judgment was based on speculation that the researchers did not account for dry puffs and overheated e-liquid. Dry puff was later discounted as relevant to aldehyde production from flavorant molecules. It was also shown that, at least in the case of one specific additive (triacetin), that acrolein, acetaldehyde and formaldehyde formation was promoted by the catalytic reaction of flavorant-derived acetic acid with e-liquid solvent. An alternative to dry puff as an explanation for enhanced levels of aerosol toxicants is film boiling. Film boiling occurs when the heat transfer between submerged metal heating coils and e-liquid is inhibited by formation of an insulating thin vapor film. The film forms due to excess heat flux at temperatures above the e-liquid boiling point, thereby promoting toxicant formation from overheated e-liquid. Significantly, e-cigarette manufacturers' recommended device power levels were shown to afford heat fluxes at levels that can produce film boiling. Film boiling thus can potentially explain why recommended safe power settings can lead to elevated aerosol toxicant emissions.In the studies described herein, a dab platform was used in conjunction with programmed temperatures and a quartz surface. There were no solvent or wicking effects involved as when using e-cigarettes, thus reducing the likelihood dry puff or film boiling as causes of ketene production. Another potential explanation for ketene as well as other toxicant emissions at relatively low temperatures is catalysis. For example, in 2018, Saliba et al. showed that toxic aldehydes, including methyl glyoxal, formaldehyde, and acetaldehyde, can form from the breakdown of the e-cigarette solvent propylene glycol at temperatures as low as ~ 80 β°C due to surface catalysis by metal heating element material (e.g., kanthal or nichrome). To investigate catalysis and ketene formation, a preliminary analysis of used cannabinoid EVALI patient vape cartridge components by Wu and O'Shea uncovered features including nickel and chromium filaments within a charred, oil-soaked, silica ceramic. Catalysis could have potentially occurred via the filaments, although charring is evidence of elevated temperatures. To the best of our knowledge, quartz (as used herein) has also not (to date) been identified as a heating component involved in catalysis during vaping. |
65b890af66c13817298d53c6 | 13 | In addition to dry puff, solvent front and catalytic effects, there is another potential cause of ketene and other toxicant emissions at relatively low vaping power settings. We first reported the effect of aerobic conditions on vaping chemistry in 2017. Upon examining the aerosol chemical profiles obtained from vaping propylene glycol and glycerol it became clear to us that oxidation reactions, particularly hydrogen atom abstractions, were prevalent during vaping. Diaz had demonstrated in 2010 that O 2 initiates the thermal degradation of propylene glycol and glycerol at significantly lower temperatures compared to anaerobic (i.e., pyrolysis) conditions. For example, O 2 -promoted hydrogen abstraction from propylene glycol was shown to occur at temperatures as low as 127 Β°C, leading to products derived from carboncentered radicals. |
65b890af66c13817298d53c6 | 14 | In our earlier studies, to confirm that O 2 promotes toxic product formation under vaping conditions, we compared product yields obtained under ambient vs. reduced-O 2 conditions. An obvious decrease in total decomposition products was observed when samples of aerosolized e-liquid were collected in a sealed glove-bag that had been flushed with N 2 . |
65b890af66c13817298d53c6 | 15 | The influence of aerobic conditions on the formation of toxic degradation products via hydrogen atom abstraction is consistent with the studies of Son and Zhao who showed that hydroxyl radicals are produced during vaping. Moreover, subsequent studies by other researchers confirmed that O 2 is a major factor in promoting aerosol toxicant formation in the presence or absence of metal catalysts, and at relatively mild vaping temperatures. The results shown in Figure embody strong evidence that ketene emissions are O 2dependent. Dabbing EtOAc as well as geranyl acetate showed clear reductions in ketene yields under anaerobic versus aerobic conditions. As noted above, theoretical studies had shown that ketene is not expected to form, from VEA or EtOAc, for example, at vaping temperatures < 700 -850 β°C, respectively. The simulated reaction pathways had activation energies of > 50 kcal/mol, which is associated with temperatures much higher than normal vaping conditions. However, the simulations were done under anaerobic (pyrolysis) conditions, which therefore can account for the discrepancies between the theoretical and experimentally observed temperatures. |
65b890af66c13817298d53c6 | 16 | In using EtOAc and geranyl acetate to compare ketene production under anaerobic and aerobic environments, the results (Figure ) showed that acetates besides phenyl acetatecontaining VEA and cannabinoids, can serve as ketene precursors. This is significant since ester flavorants are the most common class of tobacco e-cigarette flavorants. Exposure to ketene emissions may thus be more prevalent than currently known. This finding suggests the need for further studies on vaping and ketene emissions as well as ketene toxicology. |
65b890af66c13817298d53c6 | 17 | Two main issues were addressed during the studies reported herein. First, rigorous evidence for ketene formation during vaping was obtained via the use of an isotopicallylabeled acetate precursor. Second, ketene formation was shown to be feasible at common vaping temperature settings. Moreover, under anaerobic conditions, ketene formation was inhibited, demonstrating that the role of oxygen and hydroxyl radicals should be accounted for when investigating the mechanism of ketene formation. In addition, we found that ketene formation was not limited to molecules containing a phenyl acetate substructure. These findings show that theoretical models based on anaerobic pyrolysis studies may provide an insufficient estimate of the thermal oxidation chemistry that is possible under real world vaping and dabbing conditions. Further investigations into the scope of ketene formation from additional ester-containing flavorant additives is ongoing in our laboratories and will be reported in due course. prepared, the nail was heated to 378 Β°C (or 287 β°C), within the range of temperatures at which cannabis consumers typically dab. All flow rate values and puff durations were the same as previously described by us. After aerosol generation and collection over ~10 min using a CSM-STEP smoking machine, the solution was removed from the impinger and transferred to an NMR tube and immediately run on the Bruker Avance III 400 MHz NMR spectrometer. Additional trials were performed, and samples were run on the high-resolution quadrupole mass spectrometer for analysis. The samples were diluted with MeOH Anaerobic dabbing experimental setup. A Cole-Palmer glove bag (37"x37"x25") was used for all anaerobic experiments. The glove bag contained the electronic nail used in previous dabbing experiments, the dabbing glassware, and the compound of interest (for this experiment ethyl acetate or geranyl acetate was used). Three different lines were connected and sealed to the glove bag. One end of the bag was connected to the fume hood air line as well as another connection to an ultra-pure N 2 gas cylinder so switchover could occur without breaking the seal on the system. The other side of the glove bag was connected to the fume hood vacuum. Initial pressurization of the bag was achieved by closing the front zipper on the bag and taping the gas lines to ensure a proper seal. Air was allowed to flow into the bag at 2 PSI until the bag was inflated to a suitable working size. After a 5 min equilibration period the e-nail was turned on and allowed to heat to 378 Β°C. Once the desired temperature had been reached by the quartz surface (e-nail), 500 ul of ethyl acetate was administered onto the nail. The aerosol was pulled into an impinger containing the same solvent ratio as described in the section above. After 5 min the impinger was disconnected from the system and the solvent transferred to an NMR tube for analysis. After the initial samples were collected under normal atmospheric conditions, the air line was turned off and the ultra-pure nitrogen line was opened up and allowed to flow into the bag at 2 PSI while the residual air was pulled out via the fume hood vacuum at the same rate. The system was allowed to normalize to anaerobic conditions (0.0% O2 as measured by the Vzmcov 4 in 1 gas detector). Once optimal conditions were obtained the vacuum was turned off and the same dabbing experiment was again conducted under N 2 . Analysis of the impinger solvent system via NMR was again performed. |
62d6c8b2a7d17e7a5a62c38b | 0 | Metal Organic Frameworks (MOFs) are porous crystalline materials composed of metal oxide building units linked by organic linkers. The chemical diversity, especially the possibility of tunable host guest interaction, gives MOFs great potential for a wide variety of applications, not limited to energy storage, gas or molecule separation, sensing, water harvesting and purification, nano-catalysis, and drug delivery. There are a number of strategic solutions to fine-tune the physical and chemical properties of MOFs, starting from varying their building blocks, adding defects, tailoring functional groups, or by exploring other framework isomers. Isomerism is a structural phenomenon, where a chemical substance -a molecule or material has the same stoichiometry, but is different in the local structure, leading to two or more related structures. In MOFs, this condition could arise from a number of factors as classified by Zhou et al. MOFs with the same component building units but different conformation (i.e. flexible MOFs), interpenetrating structures, and MOFs with a specified topology but consisting of low symmetry building blocks, such that changing the orientation of the building block will create another isomer. [email protected] (M.A. Addicoat) ORCID(s): 0000-0003-2667-0347 (M. Nurhuda); 0000-0003-1431-6439 (Y. Hafidh); 0000-0003-1517-468X (C.C. Perry); 0000-0002-5406-7927 (M.A. Addicoat) |
62d6c8b2a7d17e7a5a62c38b | 1 | Framework isomerism is interesting to examine in detail, either considering each individual isomer or collectively, as it has been found to influence properties such as flexibility and adsorption. Conformational isomerism is reported to impact the collective flexibility in DUT-8, a MOF with Ni 2 paddlewheels, dabco pillars and naphthalene dicarboxylate (ndc) linkers. In recent work by Petkov et al., by using DFT calculations they observed the wine-rack movement of the stable isomers of DUT-8 originated from the long-range orientation of the linkers, which may point "up" or "down" relative to each paddlewheel building block. They discovered that the isomers possess different energy barriers to transform from the open form to the closed form, which results in reduced flexibility in one of the isomers. The energy barrier difference arises due to the different relative alignment of the naphthalene building blocks. |
62d6c8b2a7d17e7a5a62c38b | 2 | Wang et al., investigated the impact of ligandoriginated isomerism and ligand functionalization on gas adsorption of NbO type MOFs. Using two methoxy-functionalized diisophthalate linkers, differing in the orientation of the central part of the linker and consequently, the position of the methoxy groups, they characterise two isoreticular MOFs ZJNU-58 and ZJNU-59, which show different gas uptake and selectivity performance. The orientation of the linker in ZJNU-59 creates a narrower pore size which increases the van der Waals potential overlap thus strengthening the interaction between gas molecules and the framework. |
62d6c8b2a7d17e7a5a62c38b | 3 | One of the most frequent modifications to tune the chemistry of MOFs is linker functionalization -e.g. UiO-66, NH 2 -UiO-66, Cl-UiO-66, yet there are very limited studies on the effect of positional isomerism of these functional groups to the resulting (absorption) properties. In general functionalization adds more binding sites to the MOF structure. But adding functional groups may or may not lead to a better separation capability due to steric hindrance and / or altered pore or window size. Therefore, a methodology is needed to understand the potential diversity in positional functional group isomerism, and how these isomers affect the resultant properties of the framework molecules, eventually leading to design rules for a particular application. |
62d6c8b2a7d17e7a5a62c38b | 4 | However, addition of a simple functional group into MOF linkers creates a huge complexity in the framework, the number of isomers increasing exponentially with the number of functionalized linkers. Illustrated in Figure , suppose that the linker is a simple benzene-1,4-dicarboxylic acid (bdc), for which there are 4 hydrogen atoms where a functional group could be substituted. If the system is functionalized by one functional group per bdc linker, the number of possible isomers is 4 to the 4th power of the number of linkers, minus any duplicates arising due to symmetry of the framework. In the case of a MOF, which is typically periodic in three dimensions, considering all linkers distinctly would lead to an incredibly large number of linkers. For example, 1g of UiO-66 contains 2.171 Γ 10 21 linkers. To make this tractable, we consider isomerization at the pore level. This is justifiable as any individual guest molecule will 'see' only one pore environment at a time, and also allows application to discrete molecular cages. At the pore level then, every pore isomer is noteworthy as it has different potential surface and represents a different environment for the guest molecule, especially where the guest molecule is of sufficient size to interact with multiple functional groups on different pore walls. The absorption properties of framework materials such as MOFs are typically computed using the Grand Canonical Monte Carlo (GCMC) method. It is possible to do GCMC simulations for a small number of positional functionalization isomers, however, due to the extremely large number of possible structures the procedure needs to be incorporated into (stochastic) optimisation algorithms or machine learning approaches. Machine learning has been a very attractive research area, because it accelerates the discovery of top performing MOFs. Machine learning in general, learns patterns from provided data to make a simpler model that connects input and output. Each MOF candidate is linked to a descriptor, which can discriminate between MOFs and contains the important features that reflect the targeted property. Choosing a descriptor is the most crucial step in a machine learning procedure; if a descriptor has low correlation to the property of interest, the model will have poor predictive performance. |
62d6c8b2a7d17e7a5a62c38b | 5 | MOF descriptors that are commonly used are geometrical descriptors that describe their pore environment such as pore diameter, void fraction, framework density, volumetric and gravimetric surface area. There are also chemical descriptors to account for the different chemical environments, in a simple form they are based on atom or building block density present in the structure, or a more sophisticated descriptor is based on radial distribution functions weighted by an atomic property, such as electronegativity, polarizability and van der Waals volume for predicting gas uptake capacities. Topological descriptors account for bonding information and connectivity of atoms within a material. Some recent efforts have employed Pair Distribution Functions, PDFs, to describe MOF structure, especially where defects alter the structure. To develop our descriptor, we consider the fact that host-guest interactions occur mainly by the influence of local interaction, thereby only including a small effect from the linkers in adjacent pores. In addition, while there are 2941 3D topologies (nets) reported in the RSCR database to date, they consist of repeating a limited number of pore shapes, such as cubic, tetrahedral, octahedral, square antiprism and cuboctahedral. These two considerations combined, brings us to observe and isolate the potential created by each functionalized pore shape. To describe the distribution of chemically important functional groups, we consider only the key atoms of each functional group (e.g. the N atom of the NH 2 group) and neglect the rest of the framework, effectively resulting in a simplified PDF representing the distances between pairs of functional groups in each MOF pore. |
62d6c8b2a7d17e7a5a62c38b | 6 | Combinatorial enumeration of chemical structures, including isomers is a well-known area of chemical mathematics. . In this work, we have enumerated all possible functional group arrangements for common pore geometries, and developed a fingerprint (represented by a histogram) for characterising the dissimilarity between pore environments based on a quantification of their functional group -functional group (FG-FG) distance distribution. We propose this descriptor to be useful for molecular framework materials, such as MOFs and COFs as well as discrete porous cages such as Metal Organic Polyhedra (MOPs) and porous organic cages. |
62d6c8b2a7d17e7a5a62c38b | 7 | General pore shapes have already been listed in the context of porous organic cages. From the 20 pores (cages) identified, we focus on those having a ditopic (2connected) linker. There are 15 pore shapes that meet this criterion, as shown in Figure , we adopt the pore nomenclature and topologies from the organic cage topology classification designed by Jelfs and coworkers. The pore structures are constructed with metal nodes of radius β5Γ
, chosen to reproduce the approximate size of the Zr 6 O building block, and benzene-1,4-dicarboxylic acid (bdc) and poly(1,4-benzenedicarboxylic acid) (pbdc) for the ditopic linkers. The orientation of the benzene plane is placed such that the structure will have the highest symmetry point group. Accordingly, the hydrogen atoms on the benzene linker could be located in two distinct positions, pointing into the centre of the pore or pointing outside the pore. Topology and Nomenclature adopted from the work of Jelfs and coworkers. Upper line is the tritopic + ditopic topology family, vertices are in blue, ditopic linkers in purple. Lower line is the tetratopic + ditopic topology family, vertices are in orange, ditopic linkers in purple. Of these topologies, the π ππ‘ 2 4 π·π 8 topology is excluded from this work as no crystal structures of this topology are yet reported and the topology is not contained within the stk package. The functional group added to the linker is a Nitrogen atom (representing an amine group) with C-N bond distance 1.47Γ
and the FG-FG distance is measured between two nitrogen atoms. An example of a pore structure, π ππ‘ 2 π·π 4 , constructed using these rules is presented in Figure . The enumeration performed to the pore structure is based on 1x-functionalization or one functional group per linker. The pore structures are constructed partly using the python package supramolecular toolkit (stk). The procedure to generate and enumerate the isomers of each pore is as follows: |
62d6c8b2a7d17e7a5a62c38b | 8 | After constructing all of the pore shape structures, functionalization is applied. Every combination of functional group position in the benzene linkers are enumerated. The number of pore isomers for each topology is listed in the Supporting Information S1 However due to the orientation of the pore, some isomers could be equivalent by symmetry, thus only the unique structures are collected, while duplicates are eliminated. To examine if a structure is unique, each conformational isomer is transformed by its symmetry operations, then if an overlapping structure is found, the isomer will be identified as a duplicate, otherwise it is a unique isomer. The detailed algorithm is explained in the Supporting Information Section S2. |
62d6c8b2a7d17e7a5a62c38b | 9 | Then the total number, π of unique structures is calculated as: The histogram will show the frequency of every pair distance that exists in the pore, Figure is an example of the π ππ‘ 2 π·π 4 histogram. The sum of the frequencies of the whole histogram equals the number of FG-FG pairs, and the number of FG-FG pairs is equal to: |
62d6c8b2a7d17e7a5a62c38b | 10 | By iterating through all pairs we calculate the distance of the pair and count the frequency of the distances. When considering all the possible isomers, the histogram of FG-FG distance frequency will have larger y-values but the same distribution, with the scale of: 1 βΆ 0.0625 β
4π linker . The reason for the same distribution is because each FG position has the same occurrence in the isomer enumeration. In one isomer, the probability of occurrence of every FG position on one linker is 0.25. Therefore, the occurrence of an FG-FG pair is 0.25 Γ 0.25 which equals 0.0625. |
62d6c8b2a7d17e7a5a62c38b | 11 | The FG-FG distance frequencies are plotted as a histogram with three colour coding, representing the relative positions of the functional groups with respect to the pore, as shown in Figure . The orientation of the linker puts the functional group into two distinct positions, either pointing inside, "In" or outside, "Out" of the pore of interest. As the "In-In" pair is of most relevance to a molecule binding inside the pore, we have separated and labelled each distance with different colours. |
62d6c8b2a7d17e7a5a62c38b | 12 | The histograms of 14 pore shapes are presented in the Supporting Information Section S3 and the raw counts for both mono-functionalized bdc and pbdc linkers is included in the Supporting Information as a spreadsheet. One pore shape, π ππ‘ 2 4 π·π 8 , is excluded as no crystal structures of this topology are yet reported. Two different size of linkers are used (Figure ). The first one is the linker bdc and the second one is the pbdc linker. The π ππ‘ 2 π·π 4 pore topology has point group D 4β . The FG pair distance histogram fingerprint of the pore using linker bdc and pbdc in Figure . The blue bars in both histograms shows the distances inside the pore, and can be used to determine the size of the pore. The longest distance is approximately the cross section of the void space. Expanding the linker to pbdc, would enlarge the π ππ‘ 2 π·π 4 pore, from d = 6.0Γ
to 9.0Γ
. |
62d6c8b2a7d17e7a5a62c38b | 13 | The π ππ 4 π·π 6 pore topology has point group T π , with the total number of isomers arise from functionalization is 172. The FG pair distance histogram fingerprint of the pore using bdc and pbdc linkers in Figure . Expanding the linker to pbdc, would enlarge the π ππ 4 π·π 6 pore, from d = 6.8 Γ
to 11.8Γ
. |
62d6c8b2a7d17e7a5a62c38b | 14 | Generally changing the size of the linker to a longer linker will enlarge the void space of the pore. The histogram will consist of the same components, only the positions of each bar will be shifted. Each individual FG-FG distance is affected to a different extent by enlarging the pore (i.e. isotropic expansion of the pore does not result in even expansion of all FG-FG distances). Figure shows π ππ‘ 6 π·π 12 using linker bdc, and Figure shows π ππ‘ 6 π·π 12 using linker pbdc, the pair distances shown in green lines are the same for the two pores, however the yellow line is shifted from 6.3Γ
to 10.7Γ
. As a consequence of these different rates of distance expansion, it is possible that several FG-FG distances may merge (i.e. overlap in the histogram), or conversely, one FG-FG distance may split into several distinct distances. In this work, these effects are not seen, because the pore structures constructed using the stk program maintain the point group of the shape, thus although a substitution of a longer linker is performed, the nodes adjust to a size where the resulting structure always has the same aspect ratio. In practice, however, we expect that FG-FG distances may merge or split. An example of where this would be expected to occur is in the π ππ 2 4 π·π 6 pore topology, when the pore structure is constructed from a fixed size node and a variable size linker. Figure shows the pore structure using bdc linker, the green and the red line are at the same distance. When the linker is substituted with pbdc, the red line will expand, while the green line will remain the same, as shown in Figure . Thus the corresponding bar in the histogram will split into two. |
62d6c8b2a7d17e7a5a62c38b | 15 | Even "rigid" MOFs possess some degree of flexibility where the organic linker can rotate about the linker axis. Consequently, the distances for every pair can deviate from the original, symmetric position. To consider the effect of such linker rotation, we rotate each linker through Β±30 β’ , keeping the centre of rotation (i.e. the centre of the linker along the connector axis) fixed. For each FG-FG distance in the original descriptor, we record the minimum and maximum distance that can be attained by rotation of the linkers. Table lists the minima and maxima for each FG-FG distance in the π ππ‘ 2 π·π 4 , the π ππ 4 π·π 6 (tetrahedral), π ππ‘ 6 π·π 12 (octahedral) pores are provided in the Supporting Information Section S4. The inclusion of linker rotation to the histogram on Figure assumes no rotation barrier, addition of a rotation barrier would restrict the tails of each peak. |
62d6c8b2a7d17e7a5a62c38b | 16 | The number of possible isomers increases exponentially as the number of linker fragments comprising the pore grows. For example, the UiO-6x structure (fcu fcu fcu net) contains eight tetrahedral (π ππ 4 π·π 6 ) and four octahedral (π ππ‘ 6 π·π 12 ) pores per unit cell. A mono-functionalized π ππ 4 π·π 6 pore has 172 distinct isomers, while a similarly mono-functionalized octahedral, π ππ‘ 6 π·π 12 , pore has 354024 distinct isomers, leading to a truly staggering number of distinct structures. Yet, the chemical environment 'seen' by a guest adsorbate molecule in the pore of such a cage structure may be relatively simpler, as any given molecule is likely to only interact with a limited number of functional groups. We therefore characterise each pore environment by the pair distances between functional groups and the frequency of these distances over all possible pore isomers. |
62d6c8b2a7d17e7a5a62c38b | 17 | An efficient method to identify and count unique structures is also presented, which enables efficient generation of machine learning descriptors containing information about chemical environment provided by functionalised linkers, which is important for the calculation of absorption properties. We propose our histograms represent useful identifiers for the pore environments in MFMs that can be used to determine the adsorption of large and complex molecules in these pore environments. |
62822e8b44bdd51f26650d67 | 0 | A biosensor is an analytical device which combines a biological component with a transducer to convert a biological response into a detectable electronic signal. It has three components, the biorecognition site; the transducer; and the reader. The bio-recognition site or bioreceptor is the biological component that will interact with the analyte. This must selectively recognise the analyte of interest. Common bioreceptors include microorganisms, antibodies, enzymes, cells, and DNA. An immunosensor is a biosensor which exploits the highly specific antibody/antigen interaction. Either an antigen is immobilised onto the transducer surface and detects the antibody, or the antibody is the bioreceptor immobilised on the transducer and it detects the antigen. The interaction between the antigen and antibody cause a variation in electric charge, mass, optical or temperature properties detectable by the transducer. The transducer converts the event at the bio-recognition site into a measurable signal, which is proportional to the analyte/bioreceptor interactions. Examples of transducers include optical, electrochemical, piezoelectric and electrochemiluminescent devices. Their signal is processed by the reader and displayed in a user-friendly format. Electrochemical transducers present many advantages including their robustness, easy miniaturization, excellent detection limits, small sample volumes, ability to be used in turbid biofluids, and compatibility with integrated circuits and microfluidics. Due to these characteristics, electrochemistry devices are often adapted for pointof-care (POC) platforms. Furthermore devices can be designed to allow for simultaneous measurement of different analytes. Electrochemical impedance spectroscopy (EIS), in particular, can sensitively monitor the changes in capacitance or charge-transfer resistance associated with material binding at specifically prepared receptive electrode surfaces, and requires no prior labelling. Typically, biosensors require a label such as a fluorophore, enzyme, or nanostructure to enable detection. Labelling techniques can change binding properties of biomolecules, increase costs, assay time and requires extra sample handling making them unsuitable for POC devices. 9 |
62822e8b44bdd51f26650d67 | 1 | Antibodies, or immunoglobins (Ig), are glycoproteins produced by mammalian white blood cells, more specifically B-lymphocytes, to elicit an immune response. They bind to proteins (antigens) on the surface of pathogenic organisms e.g. bacteria, viruses, fungi, parasites, targeting them for destruction by the immune system. Antibodies recognise specific epitopes on the surface of the pathogen allowing the antibodies to either neutralise the foreign entity or facilitate further immune responses. |
62822e8b44bdd51f26650d67 | 2 | The H chain consists on one variable region V H , and three constant regions C H1 , C H2 , and C H3 . The light chains consist of one variable V L , and one constant C L region. The variable domains of the heavy and light chains form two antigen-binding sites (paratopes), creating a bivalent unit. |
62822e8b44bdd51f26650d67 | 3 | The immunoglobin monomer structure is additionally classified by its fragments. Primarily the structure is isolated into two groups, the Fc and Fab regions. The constant domain containing Fc region is the class defining region which has no antigen binding capabilities. It is in the Fab fragment that binds the antigen to the structure. The Fab comprises of one constant and one variable region from each heavy and light chain. The variable region of the antibody can also be referred to as Fv, which constitutes the paratope. |
62822e8b44bdd51f26650d67 | 4 | An impedimetric sensor is based on the EIS technique and measures the resistive or capacitive behaviour of an electrochemical system. EIS investigates the AC current response to an AC potential in respect to frequency, Ζ. A sinusoidal potential is applied to the system and the responding sinusoidal current is measured over a range of frequencies. These sinusoidal responses are at the same frequency but out of phase (Ο) with each other. The excitation voltage can be expressed as a function of time: (1) where E t is the potential at time t, E 0 is the signal amplitude and Ο is the angular frequency. The relationship between frequencies Ο (rad s -1 ) and Ζ (Hz) is: (2) The responding current can also be expressed as a function of time: (3) where I t is the current at time t, I 0 is the signal amplitude and Ο is the phase shift. A corresponding version of Ohm's law can be used to describe AC impedance: (4) This frequency-dependent impedance is expressed in terms of a magnitude, Z 0 , and phase shift. |
62822e8b44bdd51f26650d67 | 5 | Impedance is commonly described as a complex function, giving it real, Z', and imaginary, Z", components. To express impedance as a complex function Euler's relationship can be used: (5) where j is the imaginary component. The impedance can therefore be expressed in terms of complex number: (6) A Nyquist plot shows the relationship between the real and the imaginary components of impedance. |
62822e8b44bdd51f26650d67 | 6 | The imaginary axis (-Z") has a negative value as capacitance its contributing component, which always has a negative phase shift. A positive imaginary value would be due to inductance. The real axis (Z') shows the resistive contribution of the impedance. An equivalent circuit is constructed to account for the interactions at the electrode/electrolyte interface, such as the formation of the double layer or the occurrence of charge-transfer. The most commonly used circuit is the Randles circuit, which consists of solution resistance (R s ), charge-transfer resistance (R ct ), double layer capacitance (C dl ) and a Warburg element (W). The corresponding Nyquist is shown in Figure ; the semicircle data is produced by the kinetically controlled process and the diagonal line shows the effects of diffusion on the system. Alternatively, the Bode plot can be considered, for the frequency-dependency of the overall impedance or of the phase shift. By immobilising the bioreceptor to the electrode surface, the antibody-antigen binding event will create a change at the interfacial region and elicit an impedimetric response. Since EIS is sensitive to fluctuations on the electrode surface it facilitates label-free detection. To create an impedimetric immunosensor the bioreceptor must first be immobilised onto the electrode surface. To optimise the immobilisation of the bioreceptor to the electrode, the surface can first be modified. |
62822e8b44bdd51f26650d67 | 7 | However, Fab' fragments can chemisorb directly onto gold electrodes as they contain a free sulfhydryl group that forms a thiolate bond with the gold substrate, favourably orientating the antigen recognition site. To produce the Fab' fragment, the Fc region must first be cleaved with an enzyme, leaving a F(ab') 2 fragment. The disulfide bond can then be reduced to separate the two Fab' fragments, though this may also cause damage to the recognition structure as other disulfide bridges connect the heavy and light chains within the immunoglobin structure. Alternatively, for the use of whole antibodies electrode modifications are undertaken to immobilise the biorecognition element to the electrode, with improved the antibody orientation and antigen accessibility as well as increased the surface binding strength, compared to physisorption. Surface modifications must have available functional groups for linking to the biorecognition element and be stable in storage. |
62822e8b44bdd51f26650d67 | 8 | Thiols are organic compounds which are terminated by an -SH group. It is this terminal that favourably interacts with the electrode surface. The other terminal generally consists of a carboxyl or amine group for the immobilisation of biomolecules. The formation of a thiol-based SAM consists of a two-step process; the first step is the fast, physical adsorption of the thiol molecules onto the surface, with some of the molecules perpendicular to the surface. The second step is much slower as the physisorbed layer is converted to a chemisorbed monolayer and reordered to be parallel to the surface. Alkanethiols are the most established SAMs used for biosensors . Long alkyl chain thiols have a higher degree of order due to the attractive van der Waals forces between adjacent chains, and exhibits a tilt of βΌ30Β° in the direction of its nearest neighbour, suggesting longer alkanethiols (β₯10 alkyl carbons) would be advantageous for an electrode modification. However, long chains produce a densely packed monolayer which restricts access to the underlying electrode surface, inhibiting electrochemical processes. Whereas short chains have a large tilt angle and low coverage. For electrochemical sensing applications, mixed thiols are often employed, using a short chain thiol to disrupt the long chain SAM. Mixed monolayers also allow for large biorecognition elements to be sufficiently spaced apart, reducing steric hindrance, as well as allowing access for mediator molecules to the electrode surface. |
62822e8b44bdd51f26650d67 | 9 | Alkanethiol SAMs are stable at potentials between approximately 0.8 and 1.4 V versus SCE, which is compatible with most electrochemical applications. Outside of this potential window, the thiol may be oxidised or reduced, causing them to desorb from the surface. The exact potential at which this desorption occurs is dependent on the alkanethiol's structure, including the length of the alky chain. This can be used to control the formation of the SAM, as demonstrated by Satjapipat et al. Their mixed SAM consisted of 3-mercaptopropionic acid (MPA) and mercaptohexanol (MCH). |
62822e8b44bdd51f26650d67 | 10 | MPA was selectively removed by reduction desorption, resulting in bare regions on the substrate. The electrode was then placed in a solution of thiolated DNA, before the remaining adsorbed MCH could diffuse across the surface to cover the bare regions. Instead, the thiolated DNA filled these bare regions. A similar protocol could be adopted for an immunosensor using thiolated antibodies. |
62822e8b44bdd51f26650d67 | 11 | For the absorption of a mixture of two alkanethiols, the mole fraction of each thiol in solution reflects, but is not equal to the mole fraction of it as an absorbate in the SAM. Experimental conditions influence the ratio of absorbates in the SAM formed on the substrate surface. Again, the length of the alkyl chain is significant, with a bias towards the longer alkanethiol which increases over time. The choice of solvent can also be a factor if the SAM is formed from a mixture of polar and non-polar molecules. Asymmetric disulfides (RSSR') pose an alternative for forming mixed SAMs, though they have lower solubility. Their S-S bond cleaves to form a bond between the metal and the sulphur molecule (M-S), as with thiols. A similar trend is seen when using asymmetric disulfides to create a mixed SAM as with using two different alkanethiols, where there is not a 1:1 ratio of absorbates present in the SAM. |
62822e8b44bdd51f26650d67 | 12 | The longer chain tends to substitute the shorter chain due the thermodynamically favourable van der Waals interactions between the longer hydrocarbon chains. Symmetrical disulfides, such as dithiobis(succinimidyl propionate), DTSP, can also be employed for the attachment of proteins to an electrode surface. This is an attractive surface modification due to the N-hydroxysuccinimide ester terminal groups, which facilitate binding to a biorecognition element without an intermediate step or |
62822e8b44bdd51f26650d67 | 13 | cross-linker molecule. For instance, Kaushik et al. have demonstrated the use of DSH to form SAMs on a gold surface. Following the formation of a SAM layer, researchers applied the monoclonal antibodies directly onto surface to achieve their immobilisation via amide bonding. This simple and effective method was used to develop an impedimetric biosensor which targeted Ubiquitin C-terminal hydrolase L1 (UCH-L1) blood biomarker of traumatic brain injury (TBI). |
62822e8b44bdd51f26650d67 | 14 | Due to their optical properties, aromatic thiols, such as benzenethiol (BT), have become popular SAMs. SAMs formed with BT has been established as disordered, with inconsistent tilts of the phenyl ring, as both sp and sp 3 hybridised sulphur atoms existing in the monolayer. As with alkanethiols, if a long chain is substituted in the para-position, interchain interactions will increase leading to a more densely packed monolayer. Alternatively, benzenemethanethiol (BMT) which has an alkyl spacer group between the sulphur and phenyl group, forms a stable, orientated monolayer, similar to that of an alkanethiol. This is due to the favourable sp 3 hybridisation of the sulphur atom in BMT, causing a 104Β° angle with the surface to the S-C bond, respectively, and allowing a densely packed monolayer. Another common derivative of BT is aminothiophenol (ATP), which has an amine group, allowing attachment to a biorecognition element, as demonstrated by Billah et al. Vert recently, BiaΕobrzeska et al. reported the development of an impedimetric biosensor for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection. Researchers modified the gold surface with 4-ATP in order to obtain an amine functional surface. Then gluteraldehyde was used as a crosslinker to immobilise the anti SARS-CoV-2 nucleocapsid antibodies. |
62822e8b44bdd51f26650d67 | 15 | Furthermore, once 4-ATP is bound to the electrode surface, it can be electrochemically oxidised to form a radical cation, which head-to-tail couples to the neighbouring 4-ATP to 4β²-mercapto-Nphenylquinone diamine which is still bound to the surface via the terminal sulphur. Further cycling can oxidise this dimer and even hydrolyse it to form 4β²-mercapto-N-phenylquinone monoamine. Cysteamine and cystamine have also been popular monolayers for biosensor applications , despite their short carbon chain which yields insufficient interchain stabilisation. Wirde et al. found cystamine had an approximate 80% surface coverage of that for a C 18 thiol monolayer after 5 mins and with longer adsorption times this coverage increased to approach that of the long chain thiol. |
62822e8b44bdd51f26650d67 | 16 | Similarly, Briand et al. compared the immobilisation of rabbit IgG using a pure mercaptoundecanoic acid (MUA); a mixed SAM of MUA and MCH; and a SAM of cystamine. The most homogenous layer was observed for the pure MUA SAM and cystamine appeared the least homogenous. Crosslinking protocols (Section 5.1) enabled Protein A to be immobilised to the surface, which formed a homogenous layer on the pure MUA, and the cystamine SAMs, while the mixed SAM showed aggregations of Protein A. Once the capture molecule IgG was bound to the Protein A, it was found the steric hindrance caused by the closely packed cystamine layer, caused this to have the lowest antigen binding of the three SAMs. Despite these drawbacks, cystamine has successfully been utilised in biosensors as the short carbon chain molecule in a mixed SAM and in conjunction with nanoparticles. ProLinkerβ’ B is a calixarene derived, cup-shaped molecule, with two -SH groups, permitting thiol chemisorption. The biorecognition element can be immobilised directly on to the ProLinkerβ’ B layer, with favourable orientation of the antigenic binding sites. With the thiol groups interacting with the substrate surface, the ether's oxygens form the upper rim of the cup, the crown ring, locking it into a cone conformer. Due to the direction of the ProLinkerβ’ B dipole and the antibody dipole, the lowest energy and therefore most favourable orientation is an end-on interaction with the ProLinkerβ’ B. The ionised amino group from a protein has a guest-host interaction with the crown ring, with hydrophobic interactions between methoxy group of the linker and hydrophobic residues of the protein as seen in Figure . |
62822e8b44bdd51f26650d67 | 17 | "Silanes" refer to a group of compounds with four substituents on a silicon atom, customarily in the form of RSiX 3 . Though these are often described in regards to a silicon surface, they have been successfully bound to several electrode surfaces in construction of electrochemical biosensors. The three most commonly used silanes for SAM formation are 3-aminopropyltrimethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GPMS) and 3-glycidoxypropyldimethylethoxysilane (GPMES), all which contain methoxy groups to facilitate binding to the surface. Alkyltrichlorosilanes will also be discussed which have the general formula RSiCl 3 . The growth of a silane SAMs includes an irreversible covalent cross-linking step. The silicon forms bonds with the hydroxyl groups on the hydrolysed surface. The remaining Si-X bonds are hydrolysed resulting in the Si-O-Si linkage between adjacent silanes and the formation of silanols (SiOH), stabilising the layer. Water is necessary for this cross-linking step, without it the silicon will form three bonds (or however many X groups it has) to the hydroxyl groups at the surface. The most commonly used solvents are hexadecane, Isoparβ’ G and bicyclohexyl; sometimes with the inclusion of small volumes of either chloroform or carbontetrachloride to the solution to control agglomeration caused by the predominance of the cross-linking. 63 For alkyltrichlorosilanes, such as octadecyltrichlorosilane (OTS), there is a threshold temperature, below which an ordered monolayer will form and this threshold temperature is affected by the chain length. This is due to the competition between the reaction of the hydrolysed trichlorosilyl groups for the formation of a polymer or to react with the surface for the formation of a SAM. The surface reaction becomes more favourable as the temperature decreases. Biernbaum et al. compared SAMs formed from OTS and octadecyltrimethoxysilane (OTMS) and observed a difference in the tilt of the alkyl chain to the substrate surface. OTS' carbon chain was practically perpendicular to the surface (0 Β± 5Β°), whereas, OTMS has a higher tilt angle of 20 Β± 5Β°. It was proposed this was due to the different adsorption mechanisms since chlorine atoms are more reactive than methoxy groups and thus more likely to form oligomers in solution. |
62822e8b44bdd51f26650d67 | 18 | The film growth of APTES is also dependent on temperature; with dendritic islands growing and fusing to cover the surface at 10Β°C, whereas at 40Β°C a homogeneous uniform film forms. Further elevated incubation temperatures (βΌ70Β°) leads to denser, stronger and better orientated films, without compromising the self-termination of the deposition. The position of the amine group has a strong effect on its chemistry in aqueous solution, compared to silanes that lack the amine or have the amine in a different position. It has a catalytic effect on the hydrolysation of the methoxy groups and can cause the formation of a six membered hydrogen bonded ring. Howarter and Youngblood's 71 work focused on optimising the reaction conditions -temperature, concentration and time -to establish a uniform thin film. They found that the concentration of APTES solution and the temperature of the incubation governed the surface roughness and receding contact angle, whereas they had minimal effect on the film thickness. It was the incubation time that controlled this characteristic. For low concentrations (1%) good films were produced when incubation time was limited to 1 hour and these did not appear to be temperature sensitive. To produce smooth thick films at higher concentrations PAs are not typically suited to SAM formation on silicon substrates as the Si-O-P bonds are easily cleaved by nucleophiles such as water or alcohols. The T-BAG method was established to allow the chemisorption of PAs onto a silicon surface. This method forms a densely packed layer of organic groups which prohibits the diffusion of water to the interface causing the SAM to be stable against hydrolysis. Whereas, silane groups may be more suited to silicon substrates, PAs appear to be superiorly anchored to metal oxide substrates, at pH 1-10. The pH and solvent of the phosphonate solution plays an important role in the formation of a stable layer. Varying the pH and using an organic solvent has been shown to stimulate the monolayer grafting and prevent a precipitation process. Another possible issue is that the solubility will the depend on the hydrophilic/hydrophobic nature of the organic backbone, though phosphonate ester analogues are often employed as they usually show no solubility issue. As they are not as well characterised as thiols and silanes, PAs are not as often adopted as a SAM for the immobilisation of antibodies. However, both (11-hydroxyundecyl)phosphonic acid and (12carboxydodecyl)phosphonic acid were assembled on a titanium substrate and cross-linked to a protein's amino group. The hydroxyl-terminated PA was determined to form a more ordered SAM than that of the carboxyl-terminated SAM. This hydroxyl-terminated layer could be utilised for the immobilisation of antibodies or antigens to an electrode surface. |
62822e8b44bdd51f26650d67 | 19 | Polymer coatings can be applied to a variety of electrode materials but unlike alkanethiols, the absorption mechanism for polymer layers is not well understood. They can be formed on the electrode surface using two methods: solution casting of preformed polymers, or electropolymerisation of monomers. Using preformed polymers allows easy control of the particle sizes, but poor adhesion to the electrode surface. Electropolymerisation overcomes this obstacle; it involves the application of an appropriate potential to the working electrode, immersed in an aqueous solution of the monomer, allowing for better control over the deposition thickness than the casting method. Another advantage of electrodeposition is it concentrates the deposition of the polymer to the electrode area and can coat complicated geometries and 3D structures. A one-step immobilisation process can be used by including the biological component in the solution, which will be homogenously incorporated into the growing polymer, during the electropolymerisation. This entrapment of the biological molecules is popular in enzyme-based biosensors but generally is not suitable for an immunosensor. Entrapment of the molecule can diminish its biological activity due to steric hinderance, as the surrounding polymer reduces accessibility to immobilised molecule. Instead this section will focus on two-step immobilisation, where the polymer is first deposited onto the electrode surface and then the antibody or antigen is attached to the polymer layer. A two-step procedure permits the polymer to be deposited in harsher conditions for an optimal layer, while the biorecognition element can still be immobilised in mild conditions to prevent denaturing. 81 |
62822e8b44bdd51f26650d67 | 20 | Despite their insulating properties they may be used in immunosensors due to their permselectivity enhancing sensitivity and anti-fouling abilities. Polymers such as o-aminobenzoic acid (o-ABA) and o-phenylenediamine (o-PD) have been electrodeposited onto electrodes resulting in the partial insulation of the electrodes while still allowing for impedimetric response and have reducing nonspecific specific absorption onto the electrode surface. o-ABA has a carboxylic acid functional group and o-PD has an amine functional group, through which proteins can be immobilised onto the surface. |
62822e8b44bdd51f26650d67 | 21 | Chitosan is popular for electrode modifications due to its available amino groups, good adhesion and biocompatibility . For example, Soares et al. have reported a low-cost impedimetric biosensor for detection of spike protein of SARS-CoV-2 virus based on carboxymethyl chitosan which was used as a matrix for the antibodies immobilisation via carbodiimide chemistry. The developed biosensor has a limit of detection of 0.179 fg/mL for SARS-CoV-2 spike protein with high selectivity. As with o-ABA, a porous film is formed allowing reagents to reach the electrode surface, though the electrochemical response is reduced compared to the bare electrode. Non-conducting polymers are limited to thin layers to ensure it is sufficient permeable to the mediator molecule. Often conductive nanostructures are incorporated into the chitosan matrix to reduce the insulative behaviour of the coating. Luo et al. demonstrated that an electrode coated with a chitosan film containing multi-wall carbon nanotubes (MWCNT) exhibited a larger current response in 5 mM Fe(CN) 6 3-/4-than when the electrode was modified with a chitosan film without the MWCNT. Similarly, Zang et al. used a chitosan layer containing single-wall carbon nanotubes (SWCNT) to immobilise anti-aflatoxin B 1 onto the electrode surface, as chitosan enhanced the dispersion of the SWCNTs. Other groups have adopted gold nanoparticles (AuNPs), reduced graphene oxide (rGO), magnetic nanoparticles 100 and other nanoparticles 101 in conjunction with chitosan for biosensors. Electrode modification with nanostructures is discussed further in Section 4.3. |
62822e8b44bdd51f26650d67 | 22 | Conductive polymers owe their unusual electronic properties such as electrical conductivity, and high electron affinity to the conjugated Ο-electron backbone in their polymer chain. This chain has alternating single and double bonds, with sp 2 hybridised carbon atoms which generate a wide charge delocalisation. This allows charge mobility along the polymer backbone and results in metal-like semiconductor properties. Literature describes the current generate at an electrode modified with a conducting polymer and antibodies immobilised on the surface to have four steps: (i) Charge transfer at the polymer/electrode interface. |
62822e8b44bdd51f26650d67 | 23 | Common conducting polymers used in impedimetric biosensors in polypyrrole, polyaniline and polythiophene. Polypyrrole (PPy) has an advantage over other conducting polymers as it can be electropolymerised from an aqueous solution at neutral pH, whereas aniline is only soluble in acidic solutions and thiophene in organic solvents. Though thiophene is not water soluble, its derivatives can be, such as poly (3,4-ethylenedioxythiophene) (PEDOT). 107 |
62822e8b44bdd51f26650d67 | 24 | Water and/or oxygen containing solutions will experience overoxidation at lower potentials and partially deconstruct the polymer backbone, generating oxygen containing carboxyl, carbonyl, and hydroxyl groups. Although, this process reduces the conductivity of the PPy, these layers are still utilised in biosensors due to the enhanced permselectivity non-conducting polymers provide, 108 as previously mentioned. |
62822e8b44bdd51f26650d67 | 25 | Similarly, polyaniline (PANI) has multiple oxidation states referred to as leucoemeraldine, emeraldine, and pernigraniline. Leucoemeraldine, the fully reduced state and pernigraniline, the fully oxidised form, are poor conductors, even when doped with acid. Emeraldine, the half-oxidised form is considered the most useful form as it is highly stable at room temperature. It comes in two forms: |
62822e8b44bdd51f26650d67 | 26 | the emeraldine base and the emeraldine salt. The salt form which is doped with acid and the imine's nitrogen is protonated is highly conductive. The structure of the polyaniline layer can be converted between oxidation states during electrodeposition by using potentiodynamic cycling. Chen et al. 111 also produced the benzoquinone biproduct due to their potential range which induced inductive behaviour in the electrical circuit. reported an electrochemical immunosensor for hepatitis E virus detection, which was constructed on an electropolymerized PANI matrix. |
62822e8b44bdd51f26650d67 | 27 | During the electropolymerisation a polymer can be doped with counter ions, which will induce it's conductivity. The Ο-electron backbone is either oxidised (p-doping) or reduced (n-doping) and the counter anion or cation produce a neutral polymer. Some sections of the chain, referred to polarons and bipolarons, consist of radical cations or dications and act as charge carriers. The doping percentage and type of dopant will influence the level of conductivity. Possible doping ions include camphorsulphonate, tosylate, tetrafuoroborate chloride, perchlorate, sulphate and phosphate. Aniline is typically polymerised using an acid, however buffer solutions with an acidic pH have also proven to be suitable media. using phosphate buffers with pH between 1.7 and 2.2 found some phosphate species to be effective dopants. Hexacyano-ferrate (Fe(CN) 6 ) ions and ferrocene derivatives have successfully been incorporated into polymer layers. This is an attractive dopant due to excellent electrochemical behaviour due to the iron molecule which can undergo reversible electrochemical oxidation. Nanoparticles (NPs) constructed from conductive materials can also be adopted as dopants. |
62822e8b44bdd51f26650d67 | 28 | An alternative dopant method is to use a second polymer, forming a copolymer. The film properties can be regulated by adjusting the monomer concentrations. PANI co-deposited with poly(vinyl sulfonate) (PVS) has recorded improved electrode surface morphology and electrical conductivity of the polymer at non-acidic pH. 120 Poly(2-acrylamido-2-methyl propanesulphonic acid) (PAMPS) is used as a dopant to make PANI soluble in water. 121 Lignosulfonate (LGS) was shown to increase the conductivity of PANI by more than two folds as well as improving the thermal stability. 122 PANI polymers containing aniline derivatives such as o-ABA or m-aminobenzenesulphonic acid, have displayed electrochemical activity over a wide pH range, while the additional aniline increases the rate of polymerisation. Such polymers are often referred to as self-doped PANI. PPy can similarly be self-doped and be co-polymerised with pyrrole-2-carboxylic acid. As previously mentioned, these additional functional groups on the self-doped polymer films can serve as a linker for covalent attachment to a biomolecule. Polystyrene sulphonic acid (PSSA) has also successfully been incorporated into polymer layers such as PANI and PEDOT. These molecules have a higher molecular weight than counter ions and thus have a larger impact on the polymer's morphology. Additionally they are unlikely leach out or de-dope as they are partially entrapped in the layer. 103 |
62822e8b44bdd51f26650d67 | 29 | Nanostructures used in impedimetric immunosensors include metallic nanoparticles (NPs) or nanorods such as Au, Ag and Pt, as well as metal oxide NPs; magnetic NPs such as Fe 2 O 3 ; and carbon-based nanostructures such as carbon nanotubes (CNTs), carbon nanodots and graphene. As well as being used as dopants for conducting polymers, nanostructures can also be utilised to amplify the electrochemical signal by acting as a label for the target biomolecule. However, as this work focuses on label-free detection, this section will focus on nanostructures used for electrode modifications prior to biomolecule immobilisation. Nanoparticles will provide a larger surface-tovolume ratio, often leading to an increase in sensitivity, as it will increase the amount of the biorecognition element on the electrode surface. |
62822e8b44bdd51f26650d67 | 30 | Preformed NPs which are adsorbed onto the electrode surface are prepared by the reduction of metal salts in the presence of a protecting agent, such as polyvinylpyrrolidone (PVP), to prevent agglomeration. The deposition time for such NPs could be minutes to days. Electrodeposition presents a faster option for modifying an electrode. This is undertaken at a reducing potential and as with electropolymerisation the chosen parameters will affect the resulting layer. Varying applied potential or current density and deposition time will allow control of the particle size. Alternatively, NPs can be attached to the surface through a molecular linker. There are four options for this linkage as depicted in Figure , all of which require the NP to have a preformed ligand shell. The first method requires the electrode to be pre-functionalised with a SAM, which will act as the molecular linker. The terminal functional group will bind to the NP's ligand shell either through ligand exchange or electrostatic interactions. For example, a SAM layer consisting of MPA and mercaptoethylamine can be anchor Au NPs to an electrode through Au-S bond. Long chain SAMs can act as an insulative layer on the electrode, but the attachment of metallic NPs can facilitate charge transfer. The second method pre-functionalises the electrode surface with an ionically charged polymer. The NP's shell electrostatically interacts with this charged layer. Commonly used polymers for this route to capture NPs includes poly-L-lysine (PLL), poly(diallyldimethylammonium) chloride (PDADMAC), poly(amidoamine) (PAMAM), and poly-L-arginine (PLA), all of which are cationic. The third method is through "click chemistry" which coupling of the functional group on the electrode surface to the functional group on the NP ligand, such as undecyn-1-thiolate with azide through azidealkyne cycloaddition reaction to form a triazole. The fourth method directly binds the NP ligand to the electrode surface. immunosensor for the Alzheimer's biomarker detection of amyloid beta. Their SAM layer of MPA was first assembled onto the gold electrode and then Au NPs where electrodeposited onto the SAM layer using a constant potential. The AuNPs were densely and uniformly distributed, allowing for maximum antibody loading. reported origami paper based impedimetric immunosensor for C-reactive protein (CRP). For the construction of biosensor, researchers applied an electrodeposition protocol in order to obtain gold nanostructures on the electrode surface and then SAMs formation of L-Cysteine was studied onto gold nanoparticles, which was then used as an immobilisation matrix for the anti-CRP antibodies immobilisation via carbodiimide chemistry. |
62822e8b44bdd51f26650d67 | 31 | Surface functionalisation with magnetic NP Fe 2 O 3 has become prevalent due to the their low, toxicity, low cost, biocompatibility, and high electron communication. Due to their dipolar attraction leading to a tendency to agglomerate in biological solutions, chitosan is commonly used for the dispersion of these NPs on an electrode surface. Metallic coating such as Au or Ag NPs are used form a shell around magnetic beads to stabilise the them in solution and further enhance the electrochemical behaviour. The magnetic properties of Fe 2 O 3 NPs provide the unique ability to separate and isolate target molecules in biological samples through the use of an external magnet or applied magnetic field. An external magnet has also been exploited by some groups to immobilised antibody conjugated magnetic NPs onto the electrode surface. A study reported by demonstrate the use of anti-Zika virus monoclonal antibody modified magnetic nanoparticles to capture the virus particles, which are also labelled with Pt nanoparticles. After the enrichment of the virus sample, a lysis protocol was applied and the resulting sample was studied with an paper based micro chip by applying electrochemical impedance spectroscopy. Developed impedimetric biosensor based on magnetic beads separation is a highly cost-effective platform for ZIKV detection. |
62822e8b44bdd51f26650d67 | 32 | Several carbon materials have been extensively applied in the construction of biosensors as nanomaterials 148 or even standalone electrodes 149 . Among them, graphene and its derivatives are arguably one of the most popular nanostructures. Graphene is a single layer of carbon atoms organised in a sp 2 hybridised honeycomb lattice. Graphene oxide (GO) is often used instead to overcome obstacles presented by graphene, i.e. the poor production yield, requirement of surface support, and uncontrollable thickness. GO is rich in oxygen containing functional groups such as hydroxyl, epoxide, carbonyl, and carboxyl groups, which can facilitate antibody immobilisation. |
62822e8b44bdd51f26650d67 | 33 | This reduction process can be chemical, electrochemical, or thermal. There is evidence that reduction by electrochemical methods generates better electroactivity than chemical methods. This is contributed to defects produced in the GO by the electrochemical process which consequently become active sites for electroactivity . Thermal reduction has demonstrated even greater electroactivity, however the morphology of the rGO will depend on the reduction strategy and this needs to be considered when selecting a reduction method. Graphene and its derivatives have been widely used as matrixes for the development of electrochemical biosensors . Very recently, reported an impedimetric biosensor based on graphene for real-time monitoring the cytokine storm in serum which is suitable for screening COVID-19 patients. For this purpose, a graphene/copper working electrode on a sensor device was fabricated along with gold reference and gold electrodes. |
62822e8b44bdd51f26650d67 | 34 | Interestingly, the graphene electrode was not modified with any biorecognition element due to the increased variety of cytokines during the cytokine storm. However, researchers demonstrated the diagnostic capabilities of the biosensor in serum in less than 10 minutes and the biosensor was able to detect the hypercytokinemia in COVID-19 patients. reported a highly sensitive graphene based electrochemical dengue biosensor. In this study, researchers constructed the biosensor onto graphene surface, where an envelope glycoprotein domain III (cEDIII) was immobilised as biorecognition element in order to detect the anti-DENV antibodies. |
62822e8b44bdd51f26650d67 | 35 | CNTs are seamless, hollow tubes formed from graphene sheets. As with graphene the bonding is sp 2 , however Ο-Ο rehybridization occurs due to the curvature of the tube. This effect is stronger as the diameter of the tube decreases which creates a rich Ο-electron conjugation outside the tube inducing greater electrochemical activity. Hence, the length of the CNT governs the rate of electron transport. As with nanoparticles they offer a high surface-to-volume ratio and promote electron transfer. Due to the Ο-Ο stacking and van der Waals interactions between CNTs in aqueous solution, aggregation occurs causing non-homogenous and unstable coatings for electrodes when absorbed directly onto the surface. For this reason, dispersal of CNTs through a suitable polymer has become a popular method of functionalising the electrode surface. Molecular linkers as proposed for NPs can also be used. Unlike typical NPs, CNTs have structural heterogeneity due to the discrepancy between the properties of the wall and the edges. The orientation of the CNTs on the electrode surface will affect the rate of electron transfer. Vertically aligned structures result in better electrochemical performance, as the edge of the tubes have faster electron transfer kinetics than the wall. Gooding et al. vertically aligned CNTs on gold electrodes using a mixed SAM layer of cysteamine and MPA, along with dicyclohexylcarbodiimide. The electron transfer of these CNTs in the presence of Fe(CN) 6 3-/4 was 40 times faster than CNTs randomly dispersed onto the electrode. Another way to beneficially influence the electrochemical behaviour CNTs is to produce oxygenated carbon species at the tips of the tubes. CNTs have also been used commonly in combination with other materials such as biocompatible polymer chitosan . For example, a recent study shows the development of an immobilisation matrix layer by layer based on chitosan and carboxylated MWCNTs in order to immobilise the anti-Staphylococcus aureus antibodies onto an interdigitated electrode . Developed impedimetric biosensor was capable of detecting S. aureus from milk samples with limit of detection between 0.26 -1.8 CFU/mL. |
62822e8b44bdd51f26650d67 | 36 | nanosheets. TMDs have an MX 2 layered structure, where the M is a transition metal typically from group IV, V or VI and the X is a chalcogen (S, Se or Te), with the transition metal sandwiched between the chalcogens. There are strong covalent M-X intralayer bonds, while weak van der Waals bonds hold together adjacent layer, which can be cleaved to form nanosheets a few layers thick. Depending on the crystalline structure of the nanosheets, the electronic characteristics can be semiconducting, metallic, or superconducting. BN nanosheets consist of alternating boron and nitrogen atoms in a honeycomb lattice. Due to their hydrophobicity they are generally modified with a polymer such as chitosan or PDADMAC to increase their solubility and stability in aqueous solutions before immobilisation of the bioreceptor. The polymeric g-C 3 N 4 is a nitrogen doped graphene with tris-triazane based units and acts as a semiconductor. Nitrogen is a strong electron donor, changing the electronic structure from graphene, resulting in enhanced electron activity. For instance, the nitrogen atoms disrupt the basal surface, edge plane-like sites which are electrochemically active. The forms of electrode modifications that have been discussed can be combined resulting in a huge range of possible modifications. As mentioned in the previous section polymers are often deposited onto the electrode surface in conjunction with nanostructures, incorporating them into the polymers coating. However, nanostructures are also deposited onto electrodes that have already been modified with a polymer layer and form a second separate layer on top of the polymer. For instance, Narang et al. coated their impedimetric sensor with chitosan and then decorated it with Au nanorods. |
62822e8b44bdd51f26650d67 | 37 | Once the electrode has been modified the bioreceptor, i.e. antibodies or antigens, can be immobilised onto the surface. As previously mentioned, bioreceptors such as enzymes can be immobilised onto the surface during the electrode modification step, usually within a polymer layer. Although this technique is generally not adopted for immunosensors due to steric hinderance, some groups have successfully detected analytes through entrapped antibodies or antigens. John et al. deposited PPy on a platinum electrode with anti-human serum albumin (HSA). formed a semiinsulating layer on Au with insulative polymer o-phenylenediamine and antibodies for hepatic fibrosis biomarker. |
62822e8b44bdd51f26650d67 | 38 | Other electrode modifications require the bioreceptor to be immobilised after the electrode has been modified. The bioreceptor may be physiosorbed onto the modified surface, 174, 177 though this is not common due to dissociation from the surface. If the modification layer is charged it can electrostatically interact with the bioreceptor. The pH must be suitable for the bioreceptor to be charged as well. This method has drawbacks as the conformation of the protein can be changed causing denaturation; there is no control of the packing density; and there is a possibility of the biomolecules leaching. Instead, as mentioned in Section 4.1, DSP, ProLinkerβ’ B, GPMS and GPMES can directly covalently bind to the bioreceptor. Other surfaces not equipped to directly bind to the bioreceptor opt for an additional step to ensure a stable immunosensor surface. |
62822e8b44bdd51f26650d67 | 39 | These functional groups commonly terminate SAMs, side chains on a polymer backbone, or are present as ligands on nanostructures. N-hydroxysuccinimide (NHS) can activate a carboxylic acid functional via the formation of NHS-ester. Polymers can also be modified to include this NHS-ester in its structure such as pyrrole-NHS, which will directly cross-link with the bioreceptor. 178 However, NHS-esters are vulnerable to hydrolysis, thus EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) coupling is generally used instead. As seen in Figure the EDC reacts with the carboxylic acid to form an O-acylisourea intermediate. Though this intermediate can react directly with a primary amine, it is unstable and prone to hydrolysis. As hydrolysis is largely dependent on pH, an acidic buffer is used for this step. 179 NHS or its water-soluble derivative (Sulfo-NHS) couples to the carboxylate to form a more stable ester which can react with a primary amine at physiological pH. EDC also be used to covalently link phosphates to amines. 179 Electrode functionalisation may yield a surface with amines available for cross-linking. Aldehyde functional groups form imine with an amine, which can be reduced to a more stable secondary amine using NaBH 3 CN form an irreversible bond. Glutaraldehyde is a dialdehyde used to cross-link two primary amines. One aldehyde end reacts with the first amine forming and imine, leaving the other aldehyde end free to form an imine with the second amine, linking an amine on the surface to an amine on the bioreceptor. 186 |
62822e8b44bdd51f26650d67 | 40 | A non-covalent method of immobilisation exploits the high affinity proteins such as avidin, streptavidin and neutravidin have for biotin. This strong, rapidly forming non-covalent interaction is typically unaffected by pH, temperature or organic solvents. Biotin is bicyclic with a carboxylic acid group which can be functionalised to graft to the modified electrode surface. By functionalising the carboxyl group, the bicyclic ring remains intact for affinity binding. Typical biotin functionalisation includes NHS ester, or maleimide. As avidin, streptavidin and neutravidin can each bind four biotin molecules, a second biotin molecule can be conjugated to the bioreceptor. Immobilisation occurs through a biotin-avidin-biotin interaction. |
62822e8b44bdd51f26650d67 | 41 | One of the common approaches to achieve a well-oriented immobilisation of antibodies is the use of Protein G or A which are bacterial proteins from Streptococci and Staphylococcus aureus, respectively. They can be immobilised onto the modified electrode surface using cross-linking protocols discussed in Section 5.1. These proteins selectively bind to the Fc region of antibodies controlling the orientation of the bioreceptor and leaving the Fab paratope containing regions free and accessible to the antigens. Once the antibody is bound to either Protein A or Protein G it can be further stabilised by cross-linking the antibody to the Protein A/Protein G support . |
62822e8b44bdd51f26650d67 | 42 | Whereas, ProLinkerβ’ B offers a simple and rapid immobilisation procedure that does not require any chemical modification or previous manipulation of the antibodies. For example, Jarocka et al reported an electrochemical immunosensor for detection of the antibodies against influenza A virus H5N1 studied in hen serum 196 where a Protein A immobilisation was studied via carbodiimide chemistry. Then Protein A surface was used for immobilisation of anti-His antibodies and recombinant His-tagged hemagglutinin (His6-H5 HA), respectively. Another example was reported by In this study, researchers modified gold surface with Cu 2 O and Protein A, respectively, for well-oriented immobilisation of IgG anti-SARS-CoV-2 spike antibody. |
62822e8b44bdd51f26650d67 | 43 | Clearly, a biotinylated biorecognition element can attach to streptavidin modified surface site-selectively have reported an immunosensor for the impedimetric detection of myelin basic protein. For this purpose, a polyaniline layer was deposited onto electrode surface electrochemically and then the biotin was immobilised onto the polymer. Following this, biotinylated surface was treated with avidin and then biotinylated antibodies were immobilised. Developed labelless impedimetric sensor was capable of detecting the antigen within the required physiological range. |
62822e8b44bdd51f26650d67 | 44 | By modifying the electrode and immobilising biomolecules to the surface, the electrode/electrolyte interface has been altered thus changing the impedimetric response of the electrode. Formation of the antibody-antigen complex will result in a change of capacitance and electron transfer resistance at the interface, enabling detection without a label. Layer-by-layer characterisation may also be carried out as each layer added to the electrode surface will elicit a response. |
62822e8b44bdd51f26650d67 | 45 | Faradaic impedance spectroscopy monitors the change in insulation of the electrode surface in relation to a redox probe added to the electrolyte, which provides a Faradaic current. The binding of the target partial inhibits charge transfer and is observed by an increase in the real component of the impedance data i.e. R ct . A range of electrode modifications are used for Faradaic sensors including nanomaterials 204 , SAMs This layer coating the electrode must either be conductive or have sufficient gaps to allow for electron transfer. For this reason, when opting for alkanethiols, a mixed SAM layer is often chosen. SAM layers can also be coated with nanoparticles to decrease their R ct . Conductive polymers are also popular due to their unique electronic properties. |
62822e8b44bdd51f26650d67 | 46 | However, these polymers do not always produce a completely conductive layer. Strategies to induce conductivity include the incorporation of dopants, co-polymers, or conductive nanostructures during the electropolymerisation process. Often several modification methods are combined for the immobilisation of the bioreceptor to the electrode surface. For instance, some groups have decorated polymers with NPs and then coated the NPs with thiols. The equivalent circuit for such sensors is simpler than the Randles' circuit as it does not require R ct or W. Instead it considers the R s and the capacitance in the circuit. The capacitance at the electrode/electrolyte interface will consist of a multitude of capacitors connected in series. Typically, these capacitors include the capacitance of the insulating layer; the capacitance of the bioreceptor (this includes the anchoring group, along with any trapped water molecules between the protein layer and diffuse layer); and the capacitance of the bulk solution. However, if the bioreceptor does not form a completely insulating layer, resistors will be connected in parallel with the capacitor to account for the contribution of pinholes. SAMs are a popular choice for modifying the electrode surface for the immobilisation of the bioreceptor. Unlike Faradaic sensors which often used mixed SAMs, long chain alkanethiols can be ideal for capacitive sensors, as they produce dense, well-organised layer which have insulative properties. However, a second SAM molecule may be added after the original SAM formation to cover empty regions. Capacitive sensors are attractive for POC devices as no redox reagent is needed however, pinholes in the insulating layer can decrease the sensitivity of the device. Interdigitated electrodes (IDE) are made from two individually addressable comb-like structures, whose fingers interlock with a set gap between neighbouring electrodes. IDE devices have been utilised as biosensor applications, including impedimetric biosensors. They provide a larger surface area than conventional electrodes, and have a great signal-to-noise ratio which enables increased sensor sensitivity. Additionally, the geometry of the electrodes can affect the sensitivity, especially the gap between electrodes. Hence Singh et al. investigated IDEs with a constant length, and similar widths and heights, but varying nanogaps (150 to 500nm). Overall, the sensitivity of the IDE increased as the pitch decreased. A simple two-electrode set up can be employed for EIS, which along with nanoscale gaps between the electrodes, results in a small sensor footprint and makes devices compatible with mass production, which is necessary for POC devices. As with conventional single electrodes or arrays of electrodes the surface can be modified to immobilise a bioreceptor to the surface and perform label-free detection. These can be adopted as either Faradaic or capacitive devices. Generally, the biolayer is formed on both electrodes, however, as mentioned in Section 4.1.1 thiols can be selectively desorbed from the electrode surface. Applying a suitable potential to the electrode will strip the SAM layer, 233 presenting the possibility to isolate one comb to perform the assay on, or modify each comb differently. Similarly, drastically accelerated the SAM formation by applying a negative potential to one comb, reducing the deposition time of 1-dodecanethiol to 1 min. The second comb was left at open circuit during deposition and remained relatively thiol free. Alternatively, rather than modifying the electrode surface in capacitive sensors, a biolayer can be formed between the electrode gaps. As previously described, the binding of the target to the bioreceptor will push water molecules and solvated ions away from the surface. Due to the low dielectric constant of the antibody compared with water there will be a change in the dielectric properties between the electrodes causing an alteration in the capacitance, also referred to as dielectrophoretic impedance measurement. 213 |
62822e8b44bdd51f26650d67 | 47 | In the last five years many label-free impedimetric immunosensors have been developed towards POC disease diagnostics. Successful target detection includes a number of cancer biomarkers and Alzheimer disease biomarkers, and cardiovascular disease biomarkers. Diagnostics are not limited to diseases in human, pen-side devices are also desirable for animal health monitoring. Work |
62822e8b44bdd51f26650d67 | 48 | focusing on cattle has established tests for bovine IgG, liver fluke and Bovine Viral Diarrhoea. And furthermore, devices are emerging for food security, looking for contaminants such as pathogenic Escherichia coli, Ochratoxin A 252 and Aflatoxin B1. Samples for real applications in food security generally need to be centrifuged or filtered, which is currently hindering their POC implementation. Testing for contaminants in water or dairy products such as milk or infant formula proves less restrictive. An obstacle for POC electrochemical devices is the counter electrode (CE) and reference electrode (RE). Maintaining the RE and designing a suitable electrochemical cell can interfere with the portable use of devices. However, IDE allow for a two-electrode system without the need for external electrodes. Alternatively, devices are integrating the CE and RE onto the device and opting for a pseudo-reference electrode. Screen-printed electrodes are a popular option for this set up, however other nanofabrication techniques such as photo-lithography can also produce a threeelectrode system on a single substrate. Although sensors are being developed towards POC use many still have incubation times for the target β₯ 45 mins. Other groups have limited their incubation times to β€ 15 mins, which is more conducive to POC applications. In some cases, microfluidic technology is employed to decrease incubation time or facilitate analysis of multiple samples. Single frequency measurements allow monitoring of the antibody/antigen binding in real time and can help shorten the incubation time. Furthermore to develop a viable commercial product, aimed at POC diagnostics the electronic equipment required for data output must be converted into a portable format with a suitable interface to the sensor platform. Ideal this interface would be user-friendly and deliver immediate results. Zhang et al. has demonstrated Arduino board can be used as a controller unit to communicate with an impedance analyzer chip and along with a 9V battery provided the portable electronics to carry out EIS measurements on Au IDE and screen printed carbon electrodes. |
62822e8b44bdd51f26650d67 | 49 | This review highlighted methods for immobilising an antibody or antigen bioreceptor to an electrode surface for the development of an immunosensor. The focus was EIS-based sensors which facilitate label-free sensing, reducing the complexity of the assay and providing a path towards POC. Faradaic or non-faradaic modes of detection were discussed. Faradaic impedance has shown dominance in research, however non-Faradaic is employed as it does not require redox probes and is especially gaining popularity when carried out with IDEs. Immunosensors can be applied for disease diagnostics in both humans and animals, as well as in the food security sector. Recent research has shown progress in developing POC towards these sectors. Incubations times are being reduced, microfluidics offer the possibility of multiple sample testing and single frequency analysis allows for real-time monitoring of the antibody/antigen binding. |
658c9f1766c13817292b112f | 0 | increasing attention, especially in light of the urgent drive to upscale electrochemical process designs in recent years. We will focus here, however, on a considerably less studied effect of mass transport: the kinetic competition that arises from exchanging volatile reaction intermediates between the electrocatalyst surface and bulk electrolyte. This reaction model was first proposed by Behm et al. and coined the "desorption-re-adsorption-reaction" mechanism. Basis for the underlying mechanism is the desorption of a specific surface-bound and usually closed-shell reaction intermediate, whose subsequent fate creates a bifurcation in the reaction pathway: either to re-adsorb onto the surface toward full conversion, or to entirely diffuse away and thus be detected as an early partially-converted product. Electrocatalytic selectivity is thus driven by the competition between surface kinetics and diffusion. |
658c9f1766c13817292b112f | 1 | While largely overlooked, there have been some experimental reports recognizing the role of the "desorption-re-adsorption-reaction" mechanism in individual cases. In their original work, Behm et al. purposefully manipulated the catalyst loading to demonstrate how selectivity depends upon surface roughness during the eORR on Pt 17 as well as the electrochemical oxidations of methanol (eMeOHox) and ethanol (eEtOHox). More recently, similar mechanistic arguments were made to explain the varying selectivity towards CO during the eCO 2 RR on Cu when changing the catalyst morphology (via e.g. nanoparticle coverage, distance, shape, and size) or the reactant stream in reactor setups. And yet, the impact of the "desorption-re-adsorption-reaction" mechanism as well as its generalization across different processes and systems remain unclear. |
658c9f1766c13817292b112f | 2 | In this study, we demonstrate the role of mesoscopic mass transport in determining electrocatalytic selectivity for a number of technologically important processes, including the eORR and different products of eCO 2 RR on Cu. Quantitative understanding of the under-lying "desorption-re-adsorption-reaction" mechanism is established, for the first time to our knowledge, by developing a simple multi-scale model that couples diffusion to the electrochemical surface kinetics. Our model correctly reproduces a series of trends found in the experimental literature, while providing an alternative or, at least, complementary explanation to changes in electrocatalytic selectivity beyond the active site model. Within this picture, the electrode surface roughness emerges as a descriptor of selectivity that effectively captures the influence of catalyst morphology across multiple length-scales: the mesoscopic scale at the inter-particle level, microscopic scale at the intra-particle level, and nanoscopic scale at the atomic level. Our analysis finally highlights the relevance of the "desorption-readsorption-reaction" mechanism in catalyst degradation as morphological changes over time (e.g. due to nanoparticle agglomeration) induce corresponding changes in electrocatalytic selectivity. |
658c9f1766c13817292b112f | 3 | The "desorption-re-adsorption-reaction" mechanism describes the fate of a specific surfacebound, yet volatile, intermediate along the reaction path. The mechanism is schematically illustrated in Fig. where this key intermediate X * (superscript * denotes an adsorbed configuration) faces a branching in the reaction path to either (i) continue along the potentialdependent surface route towards full catalytic conversion, or (ii) desorb above the surface where it starts to build up a local concentration gradient. In the second case, if diffusion is fast enough, the near-surface species X ( * ) will leave the surface entirely and be detected as an early partially-converted product X(aq) in the bulk (aqueous) solvent. If diffusion is relatively slow, however, X ( * ) faces the prospect of re-adsorbing onto the surface where it will once again be subjected to the (i) vs (ii) competition described above. The mechanism thus essentially boils down to a competition between surface kinetics and diffusion. To describe this competition, we couple these two key components within a simple kinetic model of the selectivity-determining step. A surface-bound, closed shell intermediate X * faces the kinetic competition between a forward surface redox step vs. desorption. Following desorption, if diffusion is fast enough, near-surface X ( * ) species will escape into the bulk electrolyte and be detected as an early, partially-converted product. Our model couples diffusion with the surface kinetics via the surface roughness Ο which effectively captures the influence of catalyst morphology across multiple length-scales: at the inter-particle (via e.g. catalyst loading), intra-particle (via e.g. microscopic surface roughening), and atomic (via alloying) levels. Additionally shown are the other six parameters entering our model: free-energy barriers (βG β‘ rdx (U ), βG β‘ des , βG β‘ ads ), as well as diffusion-related properties (D diff , L diff , H s ) as described in the text. |
658c9f1766c13817292b112f | 4 | We describe the surface reaction steps through a mean-field microkinetic model, while mass transport is simply included as one-dimensional Fickian diffusion (in the direction perpendicular to the surface). The two model components are then coupled via the flux of species X ( * ) and we solve the problem iteratively for steady-state solutions. In practice, the steady-state assumption reduces the transport modeling to a simple analytical expression of Fick's first law. A converged solution is one where the diffusion flux J diff of species X ( * ) equals the corresponding flux output from the microkinetic model J mkm (=turnover frequency Γ active site density) after normalizing the rate to account for the density of catalytically active sites: |
658c9f1766c13817292b112f | 5 | Roughness thus enters the model as a central key parameter, designed to effectively capture the influence of catalyst morphology on the resulting product selectivity. Importantly, as a commonly measurable quantity (e.g. through capacitive charging, or measured current densities), this parameter Ο also allows us to draw a direct relation to experimental trends in the following. |
658c9f1766c13817292b112f | 6 | Apart from the electrocatalyst roughness, we include six additional parameters in our model. Three of these parameters enter the microkinetic model as the activation free energies for each of the competing surface reaction steps: the redox barrier βG β‘ rdx (U ) for further electrocatalytic conversion of X * at the surface, the barrier βG β‘ des for X * desorption, and the reverse barrier βG β‘ ads for X ( * ) adsorption. Among these barriers, we assume that only βG β‘ rdx depends on the applied electrode potential U , while desorption/adsorption are treated as purely 'chemical' steps and thus entirely independent of potential. This is an important distinction to make as it suggests that U can change selectivity by shifting the competition toward or against the surface redox reaction. Again, this is a dependence that we will rely upon in the following when comparing against experimental selectivity data. Finally, the remaining three model parameters are part of the transport modeling: the diffusion coefficient D diff and diffusion length L diff of species X ( * ) (which simplify the otherwise complex hydrodynamic dimensionless constants), as well as Henry's solubility constant H s which is used to convert between activity and concentration. Further details on our kinetic model can be found in the SI, Sec. S1. |
658c9f1766c13817292b112f | 7 | shows the most dramatic, exponential dependence coming from the competing reaction barriers βG β‘ rdx (U ), βG β‘ des , and βG β‘ ads , as well as from the electrode potential U (indirectly through βG β‘ rdx ). This exponential dependence follows that of the resulting reaction rates and can change the selectivity towards X(aq) from 100% to 0% within only few hundred millivolts of increasing overpotential (SI, Fig. ). In comparison, the effect from varying catalyst morphology is weaker and emerges as an asymptotic β 1/Ο dependence in selectivity. This relation is founded in the competition between diffusion and re-adsorption: the probability of X ( * ) re-adsorption increases linearly with Ο, thus leading to more of the final product and less of the volatile intermediate product. Albeit weaker than the dramatic response to U , the role of Ο can still be crucial to catalytic performance as will be demonstrated in the following. Finally, when chosen within physically reasonable ranges (SI, Fig. ), the remaining transport-related parameters (D diff , L diff , and H s ) generally influence selectivity to a much lesser extent. For example, exchanging the D diff value of acetaldehyde (13.75 x 10 -10 m 2 β’ s -1 ) to that of CO (20.3 x 10 -10 m 2 β’ s -1 ) will typically only increase selectivity towards the diffusing intermediate product by <10%. |
658c9f1766c13817292b112f | 8 | In the following, we focus on Ο and (to a second extent) U as variables against which to predict selectivity for several showcase catalytic reactions. As already indicated, the reasons for this choice include (i) the strong influence of these two parameters on selectivity with asymptotic and exponential behaviors, respectively, according to β exp (-U ) Ο , as well as (ii) |
658c9f1766c13817292b112f | 9 | the ability to draw a direct connection to measurable experimental variables. The remaining model parameters are then either taken from the literature or approximated to fit experiment (SI, Sec. S1.4). Values for the activation free energies in particular, as well the potentialdependent βG β‘ rdx (U ) functional form, are unfortunately mostly unavailable and are thus fitted to reproduce measured selectivity curves. It is important to stress, however, that the effect of Ο in these situations is always simulated by consistently re-using the same set of parameters within each reaction model studied. This allows for an unbiased comparison of the results when specifically focusing on the response to varying catalyst morphology. |
658c9f1766c13817292b112f | 10 | We first consider selectivity as a function of catalyst coverage or loading on the electrode support. This effect corresponds to catalyst roughening at the particle level and has been systematically investigated by the group of Behm for different electrocatalytic reactions on Pt electrodes. Figure shows digitized data from two such experimental studies for the (bottom). Both of these reactions involve volatile intermediates that may give rise to early, partially-converted products following the "desorption-re-adsorption-reaction" mechanism. |
658c9f1766c13817292b112f | 11 | More re-adsorption favors continued surface reaction and lowers the selectivity towards the partially-converted product. Considering its simplicity, our kinetic model captures this effect with almost remarkable agreement to experiment. The theoretical predictions (using model parameters listed in SI, Table ) are represented by the solid red lines in Fig. and show the anticipated β 1/Ο dependence discussed in the previous section. These curves largely reproduce the experimental trends. Deviation is most noticeable only under conditions of ultra-low catalyst loading (Ο < 0.2) in Fig. (a). The model's over-estimation of the experimental H 2 O 2 selectivity may be attributed to an inhomogeneous distribution of catalyst particles that cannot be described by our effective, one-dimensional Ο descriptor. The experiment-theory comparison is likely further skewed here by a more complex dependence on the applied electrode potential than that predicted by the "desorption-re-adsorptionreaction" mechanism (SI, Sec. S3.1). This potential dependence has been previously discussed for the Pt-based eORR as the result of multiple competing reaction paths (that do not go through the volatile H 2 O 2 intermediate), but such mechanistic intricacies go well beyond the scope of our simple model and are not further discussed. |
658c9f1766c13817292b112f | 12 | We next explore selectivity with varying catalyst particle shape and surface corrugation, i.e. the influence of morphological features on the mesoscopic/microscopic range. These effects are demonstrated here for the eCO 2 RR on Cu electrodes; a model system of immense technological importance due to its unique ability to synthesize both a variety of one-carbon (C 1 ), but also high-value multicarbon (C 2+ ) products. A study by the group of Jaramillo et al. identified and enumerated a total of 16 different possible products, of which we classify about half as the closed-shell intermediates that may be relevant to the "desorptionre-adsorption-reaction" mechanism. It is this intricate reaction network, along with the plethora of available experimental data, that make Cu-based eCO 2 RR an ideal playground to explore the effects of mass transport on electrocatalytic selectivity. In this section, we specifically focus on two such intermediates in the reaction mechanism: CO as the prominent first product following CO 2 electroreduction, and acetaldehyde (MeCHO) which appears further down the reaction path and competes with various other C 2+ products. Figure compiles selectivity data from different experimental studies, with very different catalyst morphologies. A Cu(111) single crystal, polycrystalline Cu foil (pc-Cu), as well as two sets of data for oxygen-derived Cu (OD-Cu) are all included in Fig. The generality of the "desorption-re-adsorption-reaction" mechanism is further strengthened by our second case study in Fig. ). This figure panel plots the selectivity towards MeCHO (vs. later C 2+ products) as a function of U SHE , while similarly compiling experimental data on different Cu morphologies: a pc-Cu sample of Ο=1, an OD-Cu sample of Ο=87, and a highly porous Cu nanoflower (Cu-flower) of Ο=390. The exponential drop of MeCHO selectivity with reducing potentials is not as clear here as in the previous example due to the limited U SHE range of the available experimental data. This relation is nevertheless predicted by our kinetic model (solid lines in Fig. )), along with the inverse relation with respect to catalyst roughness. While quantitative experiment-theory agreement is certainly far from perfect, the underlying trends are clearly compatible. Taken together, Fig. confirms the prevalent notion that copper's meso-or microscopic is critical to the resulting eCO 2 RR selectivity. These effects have so far mostly been associated, however, |
658c9f1766c13817292b112f | 13 | Our next and final case study addresses the effect of alloying. Alloying is often exploited in catalyst design as a tool that allows to manipulate the electronic properties, and hence reactivity, of active site. This concept was recently invoked to rationalize the increased selectivity towards acetate (Ac) that was measured during CO electroreduction (eCORR) on two different Cu-based alloys, namely Cu-Pd 40 and Cu-Ag. The authors of both these studies in fact found that there exists an optimal alloying ratio for which maximal selectivity towards this C 2 product is reached (SI, Sec. S3.3). They explained this on the basis of active site binding strengths. We argue here that modifying the density of active sites within the "desorption-re-adsorption-reaction" mechanism provides again an alternative, or at least complementary, reasoning to explain the reported selectivity changes. |
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