A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

High-performance amperometric biosensors and biofuel cell based on chitosan-strengthened cast thin films of chemically synthesized catecholamine polymers with glucose oxidase effectively entrapped

Rapid oxidation of dopamine (DA) or L-noradrenaline (NA) by K(3)Fe(CN)(6) yields poly(DA) (PDA(C)) or poly(NA) (PNA(C)) with glucose oxidase (GOx) effectively entrapped, and such an enzyme-entrapped catecholamine polymer is cast on an Au electrode followed by chitosan (CS) strengthening for biosensing and fabrication of a biofuel cell (BFC). The optimized glucose biosensor of CS/PDA(C)-GOx/Au displays an extremely high sensitivity up to 135 μA mM(-1) cm(-2), a very low limit of detection of 0.07 μM, a response time of <3 s, good suppression of interferents, striking thermostability (lifetime of 3 weeks at 60°C and over 2 months at 30°C), and high resistance to urea denaturation. The biosensor also works well in the second generation biosensing mode with p-benzoquinone (BQ) or ferrocene monocarboxylic acid (Fc) as an artificial mediator, with greatly broadened linear detection ranges (2.0 μM-48.0 mM for BQ and 2.0 μM-16.0 mM for Fc) and up to mA cm(-2)-scale glucose-saturated current density. The good permeability of artificial mediators across the enzyme film enables the quantification of the surface concentration of immobilized GOx on the basis of a reported kinetic model, and UV-Vis spectrophotometry is used to measure the enzymatic activity, revealing high enzymatic activity/load at CS/PDA(C)-GOx/Au. A BFC is also successfully fabricated with a bioanode of CS/PDA(C)-GOx/Au in phosphate buffer solution containing 100 mM glucose and 4.0 mM BQ and a carbon cathode in Nafion-membrane-isolated acidic KMnO(4), and its maximum power density of 1.62 mW cm(-2) is superior to those of most BFC hitherto reported.     

Urea biosensor based on PANi(urease)-Nafion/Au composite electrode

The polyaniline (PANi)-Nafion composite film was prepared onto the ceramic plate by the cyclic voltammetry (CV) method with the various cycle numbers. When the PANi-Nafion/Au/ceramic plate with the preparing cycle number of 5 was as working electrode, the cathodic peak current was achieved as 84.0 microA in 60 mg dl(-1) NH4Cl buffer solution. On the other hand, the small cathodic peak currents for buffer solution in the presence of 60 mg dl(-1) LiOH, NaCl and KCl, respectively, were found with the same composite electrode as working electrode. The cathodic peak current decreased from 84.0 to 16.3 microA in the 60 mg dl(-1) NH4Cl buffer solution when the cycle number for preparing PANi-Nafion/Au/ceramic plate composite electrode with the CV method increased from 5 to 15. The enzyme of urease was immobilized onto the PANi-Nafion/Au/ceramic plate composite film by the electrochemical immobilization and the casting methods and used as sensing electrode to detect the concentration of urea in the buffer solution. The sensitivity of composite electrode immobilized with the casting method was greater than that of electrochemical immobilization method. The sensitivity and the detecting limit of the urea sensor were found to be 0.7 and 5.27 microA (mg dl(-1))(-1)cm(-2), as well as 6 and 0.3 mg dl(-1), respectively, when urease was immobilized by glutaraldehyde (GA) cross-linker and Nafion network, respectively.     

A biofuel cell with enhanced performance by multilayer biocatalyst immobilized on highly ordered macroporous electrode

A one-compartment glucose/O(2) biofuel cell based on an electrostatic layer-by-layer (LbL) technique on three-dimensional ordered macroporous (3DOM) gold electrode was described. A 3DOM gold electrode was synthesized electrochemically by an inverted colloidal crystal template technique. Then the macroporous gold electrodes were functionalized with Au nanoparticles (AuNPs) and enzyme, glucose dehydrogenase (GDH) or laccase. The (AuNPs/GDH)(n) multilayer modified macroporous gold electrode showed excellent bioelectrocatalytic activity towards glucose. The direct electroreduction towards oxygen was realized at (AuNPs/laccase)(n) films on 3DOM gold electrodes. The maximum power density of the cell with the macroporous film as matrix was 178muWcm(-2) at 226mV, which was 16 times larger than that of the biofuel cell with the flat electrode under the same condition. The proposed method is simple and would be applicable to enhance the power output of miniaturized biofuel cell.     

A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized in a tetrathiafulvalene-tetracyanoquinodimethane/multiwalled carbon nanotubes film

A new third-generation biosensor for H(2)O(2) assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ)/multiwalled carbon nanotubes (MWCNTs) modified gold electrode. The prepared HRP/TTF-TCNQ/MWCNTs/Au electrode was used for the bioelectrocatalytic reduction of H(2)O(2), with a linear range from 0.005 to 1.05mM and a detection limit of 0.5muM for amperometric sensing of H(2)O(2). In addition, a novel method on the basis of electrochemical quartz crystal microbalance (EQCM) measurements was proposed to determine the effective enzymatic specific activity (ESA) of the immobilized HRP for the first time, and the ESA was found to be greater at the TTF-TCNQ/MWCNTs/Au electrode than that at the MWCNTs/Au or TTF-TCNQ/Au electrode, indicating that the TTF-TCNQ/MWCNTs film is a good HRP-immobilization matrix to achieve the direct electron transfer between the enzyme and the electrode.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.     

Citric acid cycle biomimic on a carbon electrode

The citric acid cycle is one of the main metabolic pathways living cells utilize to completely oxidize biofuels to carbon dioxide and water. The overall goal of this research is to mimic the citric acid cycle at the carbon surface of an electrode in order to achieve complete oxidation of ethanol at a bioanode to increase biofuel cell energy density. In order to mimic this process, dehydrogenase enzymes (known to be the electron or energy producing enzymes of the citric acid cycle) are immobilized in cascades at an electrode surface along with non-energy producing enzymes necessary for the cycle to progress. Six enzymatic schemes were investigated each containing an additional dehydrogenase enzyme involved in the complete oxidation of ethanol. An increase in current density is observed along with an increase in power density with each additional dehydrogenase immobilized on an electrode, reflecting increased electron production at the bioanode with deeper oxidation of the ethanol biofuel. By mimicking the complete citric acid cycle on a carbon electrode, power density was increased 8.71-fold compared to a single enzyme (alcohol dehydrogenase)-based ethanol/air biofuel cell.     

Electrochemical immunosensor for label free epidermal growth factor receptor (EGFR) detection

This paper presents an electrochemical immune sensor for label free detection of epidermal growth factor receptor (EGFR) by immobilizing anti-EGFR antibody (Anti-EGFRab) on dithiobissuccinimidyl propionate (DTSP) self-assembled monolayer (SAM) on gold (Au) electrode. Electrochemical studies show that increased surface concentration of redox moieties onto Anti-EGFRab/DTSP immuno-electrode leads to high electron transport and improved sensing performance. The antigen-antibody complex demonstrates a high association constant (5×10(12)L/mol) that results in high affinity of Anti-EGFRab to EGFR, confirming that the DTSP-SAM provides a conducive environment for anti-EGFR immobilization. The electrochemical response of EA/Anti-EGFRab/DTSP/Au electrode as a function of EGFR concentrations exhibits a linear range from 1pg/mL to 100ng/mL, a detection limit of 1pg/mL at a sensitivity of 2.02μAM(-1)at a regression coefficient of 0.99.     

An amperometric biosensor based on laccase immobilized onto Fe(3)O(4)NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract

A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe(3)O(4)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3V vs. Ag/AgCl. Linear range, detection limit were 0.1-10μM (lower concentration range) and 10-500μM (higher concentration range), and 0.03μM respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C.     

A comparison of different strategies for the construction of amperometric enzyme biosensors using gold nanoparticle-modified electrodes

A comparison of the analytical performances of several enzyme biosensor designs, based on the use of different tailored gold nanoparticle-modified electrode surfaces, is discussed. Glucose oxidase (GOx) and the redox mediator tetrathiafulvalene were coimmobilized in all cases by crosslinking with glutaraldehyde. The biosensor designs tested were based on the use of (i) colloidal gold (Au(coll)) bound on cysteamine (Cyst) monolayers self-assembled on a gold disk electrode (AuE) and (ii) glassy carbon electrodes (GCEs) modified with electrodeposited gold nanoparticles (nAu). The results obtained with these designs were compared with those provided by a GOx/Cyst-AuE and a GOx/MPA-AuE. In the second case (ii), configurations based on direct immobilization of GOx on nAu (GOx/nAu-GCE) or on Cyst or MPA self-assembled monolayers (SAMs) previously bound on gold nanoparticles (GOx/Cyst-nAu-GCE or GOx/MPA-nAu-GCE, respectively) were compared. The analytical characteristics of glucose calibration plots and the kinetic parameters of the enzyme reaction were compared for all of the biosensors tested. The GOx/Au(coll)-Cyst-AuE design showed a sensitivity for glucose determination higher than that achieved with GOx/Cyst-AuE and GOx/Au(coll)-Cyst/Cyst-AuE and similar to that achieved with GOx/MPA-AuE. Moreover, the useful lifetime of one single GOx/Au(coll)-Cyst-AuE was 28 days, remarkably longer than that of the other GOx biosensor designs.     

Conducting polyamic acid membranes for sensing and site-directed immobilization of proteins

A biosensor platform based on polyamic acid (PAA) is reported for oriented immobilization of biomolecules. PAA, a functionalized conducting polymer substrate that provides electrochemical detection and control of biospecific binding, was used to covalently attach biomolecules, resulting in a significant improvement in the detection sensitivity. The biosensor sensing elements comprise a layer of PAA antibody (or antigen) composite self-assembled onto gold (Au) electrode via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) linking. The modified PAA was characterized by Fourier transform infrared (FTIR), (1)H nuclear magnetic resonance (NMR), and electrochemical techniques. Cyclic voltammetry and impedance spectroscopy experiments conducted on electrodeposited PAA on Au electrode using ferricyanide produced a measurable decrease in the diffusion coefficient compared with the bare electrode, indicating some retardation of electron transfer within the bulk material of the PAA. Thereafter, the modified PAA surface was used to immobilize antibodies and then to detect inducible nitric oxide synthase and mouse immunoglobulin G (IgG) using enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and amperometric techniques. ELISA results indicated a significant amplified signal by the modified PAA, whereas the SPR and amperometric biosensors produced significant responses as the concentration of the antigen was increased. Detection limits of 3.1×10(-3)ng/ml and 2.7×10(-1)ng/ml were obtained for SPR and amperometric biosensors, respectively.     

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene-co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), CaCl₂ and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.     

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene-co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), CaCl₂ and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.     

Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics

The similar dimensions of biomolecules such as enzymes, antibodies or DNA, and metallic or semiconductor nanoparticles (NPs) enable the synthesis of biomolecule-NP hybrid systems where the unique electronic, photonic and catalytic properties of NPs are combined with the specific recognition and biocatalytic properties of biomolecules. The unique functions of biomolecule-NP hybrid systems are discussed with several examples: (i) the electrical contacting of redox enzymes with electrodes is the basis for the development of enzymatic electrodes for amperometric biosensors or biofuel cell elements. The reconstitution of the apo-glucose oxidase or apo-glucose dehydrogenase on flavin adenine dinucleotide (FAD)-functionalized Au NPs (1.4 nm) associated with electrodes, or on pyrroloquinoline quinone (PQQ)-functionalized Au NPs (1.4 nm) associated with electrodes, respectively, yields electrically contacted enzyme electrodes. The aligned, reconstituted enzymes on the electrode surfaces reveal effective electrical contacting, and the glucose oxidase and glucose dehydrogenase reveal turnover rates of 5000 and 11,800 s(-1), respectively. (ii) The photoexcitation of semiconductor nanoparticles yields fluorescence with a wavelength controlled by the size of the NPs. The fluorescence functions of semiconductor NPs are used to develop a fluorescence resonance energy transfer (FRET) assay for nucleic acids, and specifically, for analyzing telomerase activity in cancer cells. CdSe-ZnS NPs are functionalized by a primer recognized by telomerase, and this is elongated by telomerase extracted from HeLa cancer cells in the presence of dNTPs and Texas-red-functionalized dUTP. The dye integrated into the telomers allows the FRET process that is intensified as telomerization proceeds. Also, the photoexcited electron-hole pair generated in semiconductor NPs is used to generate photocurrents in a CdS-DNA hybrid system associated with an electrode. A redox-active intercalator, methylene blue, was incorporated into a CdS-duplex DNA monolayer associated with a Au electrode, and this facilitated the electron transfer between the electrode and the CdS NPs. The direction of the photocurrent was controlled by the oxidation state of the intercalator. (iii) Biocatalysts grow metallic NPs, and the absorbance of the NPs provides a means to assay the biocatalytic transformations. This is exemplified with the glucose oxidase-induced growth of Au NPs and with the tyrosinase-stimulated growth of Au NPs, in the presence of glucose or tyrosine, respectively. The biocatalytic growth of the metallic NPs is used to grow nanowires on surfaces. Glucose oxidase or alkaline phosphatase functionalized with Au NPs (1.4 nm) acted as 'biocatalytic inks' for the synthesis of metallic nanowires. The deposition of the Au NP-modified glucose oxidase, or the Au NP-modified alkaline phosphatase on Si surfaces by dip-pen nanolithography led to biocatalytic templates, that after interaction with glucose/AuCl4- or p-aminophenolphosphate/Ag+, allowed the synthesis of Au nanowires or Ag nanowires, respectively.     

Amperometric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) film

Organic peroxides, t-butyl hydroperoxide, 2-butanone peroxide, cumene hydroperoxide and t-butyl peracetate, were determined by an amperometric enzyme electrode. The enzyme electrode was prepared through electrostatic immobilization of horseradish peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF(+)ClO(4)(-) film was coated on a Pt foil at +0.70 V by electrooxidation of polyvinylferrocene in methylene chloride with 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode PVF(+)HRP(-) was prepared by anion-exchange in a solution of HRP(-) in 0.05 M phosphate buffer at pH 8.5. FTIR spectroscopy was used to identify PVF, PVF(+)ClO(4)(-), and PVF(+)HRP(-). The immobilized amount of the enzyme in the film was determined by UV spectroscopy. The effects of the polymeric film thickness, bulk enzyme concentration used in the immobilization treatment and the temperature on the performance of enzyme electrode were investigated. The inhibitory effect of oxygen was also examined. Linearities, lower detection limits, active life times and sensitivities of the electrode were determined for each peroxide.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

Exogenous ABA induces salt tolerance in indica rice (Oryza sativa L.): The role of OsP5CS1 and OsP5CR gene expression during salt stress

Abscisic acid (ABA) applied exogenously at 100 μM prior to and during the salt-stress period induced salt tolerance in both the salt-susceptible (LPT123) and the genetically related salt-resistant (LPT123-TC171) rice lines, enhanced the survival rate by 20%, and triggered proline (Pro) accumulation earlier than that by salt-stress alone, supporting a role for Pro as an osmoprotectant. In both rice lines, salt-stress induced OsP5CS1 gene expression, suggesting that proline accumulation occurs via OsP5CS1 gene expression during salt stress. An increase in the endogenous ABA level was required for the induction of OsP5CS1 gene expression by salt stress. Under salt stress, topical ABA application-induced OsP5CS1 gene expression only in the salt-resistant line but up-regulated OsP5CR gene expression in both rice lines, suggesting that the increased proline accumulation and salt resistance induced by topical ABA application may result from the up-regulation of OsP5CR and not, directly at least, from OsP5CS1. Moreover, exogenous ABA application up-regulates OsCam1-1 (the salt-stress-responsive calmodulin) gene expression, and calmodulin was shown to play a role in the signal transduction cascade in proline accumulation during salt stress. These data suggest the role of the calmodulin signaling cascade and the induction of OsP5CR gene expression in proline accumulation by exogenous ABA application.