it is widely considered meaningful to develop new selective and sensitive chemosensors for the detection of metal ions due to their important roles in medicine , living systems , and the environment.1,2 among metals , aluminum is the third most prevalent element ( 8.3  % by weight ) in the earths crust . acid rain can leach aluminum from soil and increases the concentration of free al in surface water , which is detrimental to growing plants.35 consequently , about 40  % of the worlds acidic soil is caused by aluminum toxicity.68 furthermore , people are widely exposed to aluminum because of its widespread use in food additives , aluminum - based pharmaceuticals , and so on.9,10 the toxicity of aluminum leads to damage to the central nervous system ( cns ) , which is suspected to be involved in neurodegenerative diseases such as alzheimers and parkinsons diseases.11,12 the world health organization ( who ) reported that the average daily human intake of aluminum is approximately 310  mg.13,14 however , compared with other metal ions , the detection of al has always been a great challenge because of its poor coordination ability , strong hydration ability , and the lack of spectroscopic characteristics.1517 to date , many fluorescent probes have been developed to detect al.1832 to the best of our knowledge , although many schiff base derivatives bearing a fluorescent moiety have been used to detect various metal ions,33 schiff base - type al sensors used for bioimaging remain rare.20,28,30,32d here , we report a previously identified schiff base derivative ( 1)34 ( scheme 1 ) as the fluorescent turn - on probe for detection of al . compared with the reported probes for al , probe 1 can be synthesized easily in high yields . common coexistent metal ions showed little or no interference in the detection of al , demonstrating the high selectivity of probe 1 . more importantly , the detect limit of probe 1 towards al is extremely low ( 6.710  m ) , suggesting its high sensitivity . therefore , the selectivity of 1 to various metal ions , namely k , co , ca , ba , ni , pb , hg , ce , zn , cd , fe , al ( 5.0  equiv ) , was examined in an acetonitrile / water solution ( 1:1 v / v ) . as shown in figure  1 , probe 1 alone displays very weak fluorescence when excited at 400  nm . the addition of 5.0  equiv of k , co , ca , ba , ni , pb , hg , ce , zn , cd , or fe had no obvious effect on the fluorescence of probe 1 . however , upon addition of 5.0  equiv of al , the emission intensity was largely enhanced at 467  nm . fluorescence responses of 1 ( 5  m , ex=400  nm ) upon addition of 5.0  equiv of various metal ions in ch3cn / h2o ( 1:1 v / v ) . achieving a highly selective response to the targeted analyte over a complex background of potentially competitive species is an important requirement for a chemosensor . thus , the competitive experiments were then carried out by addition of 10.0  equiv of a to the solutions of 1 in the presence of 100.0  equiv of other metal ions , in order to validate the high selectivity of 1 as a chemosensor for the detection of al . as shown in figure  2 , there is nearly no interference for the detection of al in the presence of other metal ions . these results demonstrate that 1 displays a high selectivity for al in the acetonitrile / water solution ( 1:1 v / v ) . fluorescence responses of 1 ( 5  m ) at 467  nm , in the absence and presence of different metal ions ( 5.0  equiv ) . the measured fluorescence intensity ( i  i0 ) of probe  1 in the presence of both the indicated metal ion ( black bar ) and the indicated metal ion plus 100  equiv al ( red bar ) . the uv - vis absorption of 1 in acetonitrile / water ( 1:1 v / v ) was investigated by spectrophotometric titration . as shown in figure  3  a , in the presence of al ( 5.0  equiv ) , a new absorbance band appears at approximately 350  nm . however , no obvious changes in the uv - vis absorption spectra of 1 were observed in the presence of other metal ions . furthermore , the absorbance peak at 350  nm increased gradually upon gradual addition of al to the solution of 1 ( figure  3  b ) . a )  uv - vis absorption spectra of 1 upon addition of various metal ions ( 5.0  equiv ) in ch3cn / h2o ( 1:1 v / v ) ; b )  absorption titration spectra of 1 ( 5  m ) in ch3cn / h2o ( 1:1 v / v ) in the presence of increasing concentrations of al ( 08  equiv ) . titration of a 5  m solution of 1 in acetonitrile / water ( 1:1 v / v ) by gradual addition of al was carried out . upon incremental addition of al , the fluorescence intensity at 467  nm of 1 in solution figure  4  b shows relative intensity ( i / i0 ) versus the concentration of al in the low concentration region up to 6.710  m , suggesting that probe 1 could act as a chemosensor for the detection of al with extraordinarily high sensitivity compared with other chemosensors.1832 a )  fluorescence titration of 1 ( 5  m ) with al in ch3cn / h2o ( 1:1 v / v ) with increasing concentrations of al ( ex=400  nm ) ; b )  fluorescence of 1 ( 5  m ) at 467  nm as a function of al concentration ( 0810  m ) . to investigate the binding stoichiometry of the 1al complex , a jobs plot based on fluorescence data was generated ( figure  5 ) , showing a maximum emission intensity when the molecular fraction of al was approximately a half , suggesting that probe 1 complexes with al in a 1:1 ratio . according to the linear benesi i0 ) at 467  nm showed a linear relationship with /[al ] . using the benesi  hildebrand plot ( figure  6 ) , the binding constant of the 1al complex was found to be 4.2510  m ( r=0.998 ) . jobs plot to determine the stoichiometry of the 1al complex ( ex=400  nm ) . fluorescence at 467  nm was plotted as a function of the molar ratio : [ al]/([al]+ ) . i0 ) of 1 as a function of 1/[al ] . the ph influence on the sensitivity of probe 1 for al was also examined under various ph conditions ( figure  7 ) . as expected , 1 was found to be weakly fluorescent in acetonitrile / water ( 1:1 v / v ) and showed a significantly positive response ( strong fluorescence ) between ph  6.8 and 7.4 upon the addition of al . the biologically relevant ph range is 6.07.6,20 thus , fluorescent probe 1 could be used to detect intracellular al without being affected by the physiological ph . effect of ph on the fluorescence intensity of chemosensor 1 ( 5  m ) (  ) at 467  nm , and 1 upon addition of 10  equiv al in ch3cn / h2o ( 1:1 v / v ) (  ) . the ability of biosensing molecules to selectively monitor guest species in living systems recently attracted much attention due to their importance in biological applications.36,37 therefore , we evaluated fluorescent probe 1 against escherichia coli , a gram - negative bacteria , to demonstrate its potential application in biological systems . e.  coli cells were incubated with 1 ( 10  m ) for one hour at 37  c , after which time no obvious fluorescence could be detected ( figure  8  a ) . in contrast , a strong fluorescent image was observed in e.  coli cells incubated with both 1 and al ( figure  8  b ) . the bright field transmission image of the e.  coli cells ( figure  8  c ) was exactly consistent with the fluorescence image in figure  8  b. these results suggested that 1 is cell - membrane permeable and could be used for detecting al within living systems . a )  bacteria after incubation with probe 1 ( 10.0  m ) for 1  h ; b )  bacteria after incubation with alcl3 ( 200  m ) in dmso / h2o ( 1:1 v / v ) for 10  min , then with 1 ( 10.0  m ) for 1  h ; c )  bright field image of e.  coli . the images were taken at 2000 magnification , and the size of each square presented is 25  m25  m . we have synthesized a probe ( 1 ) that could selectively detect al with high sensitivity . upon the formation of the 1al complex , obvious detectable changes in absorbance and fluorescence were observed . the recognition behavior of probe 1 towards al was carefully investigated by experimental studies to give the binding stoichiometry and binding constant , and the level of detection limit , which is extremely low . more importantly , confocal laser scanning microscopy showed the application of fluorescent probe 1 as an imaging reagent for detection of al in e.  coli , demonstrating its value in practical applications in biological systems . general materials and methods : all materials for synthesis of probe  1 were obtained in analytical grade from alfa aesar , a johnson matthey company , and used without further purification . nmr spectra were recorded on a bruker drx spectrometer operating at 500  mhz or 125  mhz ( bruker scientific technology co. ltd , beijing , p. r. china ) . chemical shifts (  ) are reported in parts per million ( ppm ) relative to an internal standard ( tms ) . a hitachi f-4500 spectrofluorometer ( hitachi high technologies co. ltd , shanghai , p. r. china ) was used for fluorescence measurements . absorption spectra were recorded with a techcomp uv-8500 spectrophotometer ( shanghai techcomp scientific instrument co. ltd , shanghai , p. r. china ) . n-(4-diethylamino-2-hydroxybenzylidene)-2-hydroxybenzohydrazide ( 1):34 a solution of 2-hydroxybenzohydrazide ( 3 ) ( 1.52  g , 10  mmol ) , 4-(diethylamino)salicylaldehyde ( 2 ) ( 1.93  g , 10  mmol ) , and piperidine ( 0.50  ml ) in etoh ( 25  ml ) was heated at reflux for 4  h. the resulting yellow precipitate was isolated by filtration and then washed with etoh to give the crude product . purification by column chromatography ( hexane / etoac , 2:11:1 ) gave the title compound as a pale yellow solid ( 2.77  g , 81  % yield ) : h  nmr ( 500  mhz , [ d6]dmso ) : =8.48 ( s , 1  h ) , 7.87 ( d , j=6.5  hz , 1  h ) , 7.447.42 ( m , 1  h ) , 7.29 ( d , j=8.5  hz , 1  h ) , 6.976.93 ( m , 2  h ) , 6.38 ( d , j=8.5  hz , 1  h ) , 6.24 ( s , 1  h ) , 3.38 ( q , j=7.0  hz , 4  h ) , 1.09  ppm ( t , j=7.0  hz , 6  h ) ; c  nmr ( 125  mhz , [ d6]dmso ) : =165.0 , 159.8 , 159.6 , 150.2 , 134.5 , 132.1 , 129.2 , 119.5 , 117.8 , 116.0 , 48.5 , 11.7  ppm ; ms ( esi ) : m / z=327 [ m ] . fluorometric and uv - vis titrations : solutions ( 5.0  mm ) of k , co , ca , ba , ni , pb , hg , ce , zn , cd , fe , and al in ch3cn / h2o ( 1:1 v / v ) were prepared from the chloride or nitrate salt . a solution ( 5  m ) of probe  1 in ch3cn / h2o ( 1:1 v / v ) was also prepared for titration purposes . , 5.0  mm metal ion solution was added using a micro - injector to a solution of 1 ( 5  m , 3  ml ) ; the volume of metal ion solution could be ignored compared to that of 1 . measurements were taken after 2  h. excitation and emission slits of 5.0  nm were used for all the measurements of fluorescence . preparation of e.  coli cells : e.  coli was obtained from the qingdao institute of bioenergy and bioprocess technology ( qibebt ) of the chinese academy of sciences ( cas ) . nutrient broth and trypticase soy broth were obtained from aladdin reagent company ( shanghai , china ) . after incubation at 37  c for 24  h , stock cultures were maintained on trypticase soy agar slants at 4  c . a loop of bacteria was resuspended in nutrient broth ( 5  ml ) and trypticase soy broth ( 50  ml ) without glucose ( 1.5  % trypticase , 0.5  % soytone , and 0.5  % nacl ) . the culture broth was incubated with shaking for 1518  h at 37  c . bacteria were washed with sterile 0.85  % saline ( 45  ml ) and then centrifuged twice ( 3000  rpm , rt , 30  min ) to remove culture medium from the bacterial pellet . before use , bacteria were resuspended in 0.025  m phosphate buffer ( ph  6.8 ) ( 4  ml ) . confocal fluorescence imaging : confocal fluorescence imaging was performed on a leica tcs sp5 laser scanning confocal microscope ( leica microsystems , wetzlar , germany ) , with a blue diode laser ( 50  mw , ex=405  nm ) and acquisition as xyz at a speed of 400  hz . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .
the development of novel selective probes with high sensitivity for the detection of al3 + is widely considered an important research goal due to the importance of such probes in medicine , living systems and the environment . here , we describe a new fluorescent probe , n-(4-diethylamino-2-hydroxybenzylidene)-2-hydroxybenzohydrazide ( 1 ) , for al3 + . 
 probe 1 was evaluated in a solution of acetonitrile / water ( 1:1 v / v ) . 
 compared with previously reported probes for al3 + , probe 1 can be synthesized easily and in high yield . 
 a job plot confirmed that probe 1 is able to complex al3 + in a 1:1 ratio , and the binding constant was determined to be 4.25108  m1 . 
 moreover , the detection limit was as low as 6.7109  m , suggesting that probe 1 has a high sensitivity . 
 common coexistent metal ions , such as k+ , co2 + , ca2 + , ba2 + , ni2 + , pb2 + , hg2 + , ce2 + , zn2 + , cd2 + , fe3 + , showed little or no interference in the detection of al3 + in solution , demonstrating the high selectivity of the probe . finally , the ability of probe 1 to act as a fluorescent probe for al3 + in living systems was evaluated in gram - negative bacteria , escherichia coli , and confocal laser scanning microscopy confirmed its utility . 
 the results of this study suggest that 1 has appropriate properties to be developed for application as a fluorescent probe of al3 + for use in biological studies .