Impedimetric biosensor based on cell-mediated bioimprinted films for bacterial detection

This work presents the synthesis of bacteria-mediated bioimprinted films for selective bacterial detection. Marine pathogen sulfate-reducing bacteria (SRB) were chosen as the template bacteria. Chitosan (CS) doped with reduced graphene sheets (RGSs) was electrodeposited on an indium tin oxide electrode, and the resulting RGSs-CS hybrid film served as a platform for bacterial attachment. The electrodeposition conditions were optimized to obtain RGSs-CS hybrid films with excellent electrochemical performance. A layer of nonconductive CS film was deposited to embed the pathogen, and acetone was used to wash away the bacterial templates. Electrochemical impedance spectroscopy was performed to characterize the stepwise modification process and monitor the SRB population. Faradic impedance measurements revealed that the charge transfer resistance (R(ct)) increased with increased SRB concentration. A linear relationship between ΔR(ct) and the logarithm of SRB concentration was obtained within the concentration range of 1.0×10(4)cfumL(-1) to 1.0×10(8)cfumL(-1). The impedimetric sensor showed good selectivity towards SRB based on size and shape. Hence, selectivity for bacterial detection can be improved if the bioimprinting technique is combined with other bio-recognition elements.     

Direct immobilisation of antibodies on a bioinspired architecture as a sensing platform

A sensitive and selective immunosensor for the nonlabeled detection of sulfate-reducing bacteria (SRB) is constructed using a self-polymerised polydopamine film as the immobilisation platform. Self-polymerisation of dopamine is used as a powerful approach for applying multifunctional coatings onto the surface of a gold electrode. The polydopamine film is used not only as the immobilisation platform, but also as a cross-linker reagent for the immobilisation of the anti-SRB antibody. The polydopamine film is loaded with a high density of anti-SRB antibodies linked to the substrate to obtain high response signals. The formation and fabrication of the biosensor and the quantification of antibody anchoring are monitored, and SRB detection is performed by either quartz crystal microbalance (QCM) or electrochemical impedance spectroscopy (EIS). After modeling the impedance Nyquist plots of the SRB/anti-SRB/polydopamine/gold electrode for increasing concentrations of SRB, the electron transfer resistance (R(ct)) is used as a measure of immunocomplex binding. The R(ct) is correlated with the concentration of bacterial cells in the range of 1.8×10(2) to 1.8×10(6) CFU mL(-1); the detection limit is 50 CFU mL(-1). This work demonstrates a new immobilisation platform for the development of a sensitive and label-less impedimetric and piezoelectric immunosensor. This immunosensor may be broadly applied in clinical diagnoses and the monitoring of water environmental pollution. The method proposed is distinct in its ease of application, use of a simple protocol, and mild reaction conditions. These allow it to be applied to a wide variety of materials.     

Construction of an amperometric glucose biosensor based on the immobilization of glucose oxidase onto electrodeposited Pt nanoparticles-chitosan composite film

One-step construction of Pt nanoparticles-chitosan composite film (PtNPs-CS) was firstly proposed as a novel immobilization matrix for the enzymes to fabricate glucose biosensor. This novel interface embedded in situ PtNPs in CS hydrogel was developed by one-step electrochemical deposition in solution containing CS and chloroplatinic acid (H(2)PtCl(6)). Several techniques, including scanning electron microscopy (SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronoamperometry were employed to characterize the assembly process and performance of the biosensor. Under the optimized experimental conditions, the resulting biosensor exhibited excellent linear behavior in the concentration range from 1.2 μM to 4.0 mM for the quantitative analysis of glucose with a limit of detection of 0.4 μM at a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 2.4 mM, showing good affinity. The proposed biosensor offered good amperometric responses to glucose due to the nanostructured sensing film provided plenty of active sites for the immobilization of glucose oxidase (GOD).     

Impedance studies of a nano-structured conducting polymer and its application to the design of reliable scaffolds for impedimetric biosensors

Electrochemical impedance spectroscopy (EIS) as a powerful, non-invasive and informative technique was used to obtain important information about kinetics of doping process in conducting polymers. Polypyrrole (PPy) and its derivatives can form conducting polymer films which represent excellent charge transfer behaviors during doping processes. It can also have a wide range of applications in bioelectrochemistry. In the present study the conducting polymer of alpha-carboxy pyrrole (alpha-COOH-PPy), appended onto the underlying film of PPy, was prepared by electrochemical methods and its behavior was analyzed using EIS. From highly accurate fitting of impedance results it was found that the charging mechanism is governed by the diffusion process. In addition, the impedance analyses provided values for the bulk polymer parameters including diffusion coefficient (D), equilibrium capacitance (C(0)) and diffusion resistance (R(0)). The surface morphology of the polymeric film was characterized using scanning electron microscopy (SEM). The film was then used to immobilize the cytochrome C (cyt-C) and to perform its electrochemical studies. The modified cyt-C/alpha-COOH-PPy electrode was used for electrocatalytic reduction of H(2)O(2) in solution and its viability as a new impedimetric biosensor was examined. Based on the calibration curve obtained for the proposed impedimetric biosensor, the limit of detection and relative standard deviation were evaluated as 0.25mumolL(-1) and 7%, respectively. Finally, the prolonged stability test was performed and high stability and reproducibility of the new biosensor was confirmed.     

DNA electrochemical biosensor based on thionine-graphene nanocomposite

A novel protocol for development of DNA electrochemical biosensor based on thionine-graphene nanocomposite modified gold electrode was presented. The thionine-graphene nanocomposite layer with highly conductive property was characterized by scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. An amino-substituted oligonucleotide probe was covalently grafted onto the surface of the thionine-graphene nanocomposite by the cross-linker glutaraldehyde. The hybridization reaction on the modified electrode was monitored by differential pulse voltammetry analysis using an electroactive intercalator daunomycin as the indicator. Under optimum conditions, the proposed biosensor exhibited high sensitivity and low detection limit for detecting complementary oligonucleotide. The complementary oligonucleotide could be quantified in a wide range of 1.0 × 10(-12) to 1.0 × 10(-7)M with a good linearity (R(2)=0.9976) and a low detection limit of 1.26 × 10(-13)M (S/N=3). In addition, the biosensor was highly selective to discriminate one-base or two-base mismatched sequences.     

A new impedimetric biosensor utilizing VEGF receptor-1 (Flt-1): early diagnosis of vascular endothelial growth factor in breast cancer

A new impedimetric biosensor, based on the use of vascular endothelial growth factor receptor-1 (VEGF-R1), was developed for the determination of vascular endothelial growth factor (VEGF). VEGF-R1 was immobilized through covalent coupling with 3-mercaptopropionic acid which formed a self-assembled monolayer on gold electrodes. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy techniques were employed to characterize the immobilization process and to detect VEGF. To successfully construct the biosensor current, experimental parameters were optimized. Kramers-Kronig Transform was performed on the experimental impedance data. The obtained results provided a linear response range from 10 to 70 pg/mL human VEGF. The applicability of the developed biosensor in the determination of VEGF in a spiked artificial human serum sample was experienced, yielding average recovery of 101%, in that order, with an average relative deviation value less than 5%.     

Electrochemical sensing of trimethylamine based on polypyrrole-flavin-containing monooxygenase (FMO3) and ferrocene as redox probe for evaluation of fish freshness

Amperometric and impedimetric biosensor for detecting trimethylamine (TMA) which represents good parameters for estimating fish freshness has been developed. The biosensor is based on a conducting polypyrrole substituted with ferrocenyl, where flavin-containing monooxygenase 3 (FMO3) enzyme was immobilised by covalent bonding. FMO3 catalyzes the monooxygenation TMA to trimethylamine N-oxide (TMO). For catalysis FMO require flavin adenine (FAD) as a prosthetic group, NADPH as a cofactor and molecular oxygen as cosubstrate. Ferrocenyl group substituted on the polypyrrole matrix will serve as redox probe for monitoring the response of the biosensor to TMA. The construction of the biosensor was characterized by FT-IR, cyclic voltammetry and impedance measurements. Detection is done through the analysis of the current of oxidation signal of the ferrocenyl groups and compared to the measurement of impedance related to the electrical properties of the layers. Amperometric and impedimetric response were measured as a function of TMA concentration in range of 0.4 μgm L(-1)-80 μgm L(-1) (6.5 μmol L(-1)-1.5 mmol L(-1)). Amperometric measurements show a decrease in current response which is in correlation with the increase of the charge transfer resistance demonstrated by impedance. Calibration curve obtained by impedance spectroscopy shows a high sensitivity with a dynamic range from (0.4 μgm L(-1) to 80 μgm L(-1)). We demonstrated, using ferrocene as redox probe for catalytic reaction of FMO3, that high sensitivity and dynamic range was obtained. The biosensor was stable during 16 days. The biosensor shows high selectivity and its sensitivity to TMA in real samples was evaluated using fish extract after deterioration during storage.     

Electrochemical impedimetric immunosensor for insulin like growth factor-1 using specific monoclonal antibody-nanogold modified electrode

An electrochemical impedimetric immunosensor was developed for ultrasensitive determination of insulin-like growth factor-1 (IGF-1) based on immobilization of a specific monoclonal antibody on gold nanoparticles (GNPs) modified gold electrode. Self-assembly of colloidal gold nanoparticles on the gold electrode was conducted through the thiol groups of 1,6-hexanedithiol (HDT) monolayer as a cross linker. The redox reactions of [Fe(CN)(6)](4-)/[Fe(CN)(6)](3-) on the electrode surface was probed for studying the immobilization and determination processes, using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The interaction of antigen with grafted antibody recognition layer was carried out by soaking the modified electrode into antigen solution at 37°C for 3 h. The immunosensor showed linearity over 1.0-180.0 pg mL(-1) and the limit of detection was 0.15 pg mL(-1). The association constant between IGF-1 and immobilized antibody was calculated to be 9.17×10(11) M(-1). The proposed method is a useful tool for screening picogram amounts of IGF-1 in clinical laboratory as a diagnostic test.     

Label-free impedimetric immunosensor for ultrasensitive detection of cancer marker Murine double minute 2 in brain tissue

The detection of cancer biomarkers is as important tool for the diagnosis and prognosis of cancer such as brain cancer. Murine double minute 2 (MDM2) has been widely studied as prognostic marker for brain tumor. Here we describe development of a new sensitive label free impedimetric immunosensor for the detection of MDM2 based on cysteamine self assembled monolayers on a clean polycrystalline Au electrode surface. The amine-modified electrodes were further functionalized with antibody using homobifunctional 1,4-phenylene diisothiocyanate (PDITC) linker. The assembly processes of the immunosensor had been monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques using Fe(CN)(6)(3-/4-) solution as redox probe. The impedance changes upon binding of MDM2 protein to the sensor surface was utilized for the detection of MDM2. The increase in relative electron-transfer resistance (ΔR/R(0)%) values was linearly proportional to the concentration of tumor marker MDM2 in the wide dynamic range of 1pg/ml-1μg/ml. The limit of detection was 0.29pg/ml in phosphate buffer saline (PBS) and 1.3pg/ml in mouse brain tissue homogenate, respectively. The immunosensor showed a good performance in comparison with ELISA for the analysis of the MDM2 in the cancerous mouse brain tissue homogenates. Moreover, the immunosensor had a good selectivity against epidermal growth factor receptor (EGFR) protein, long-storage stability and reproducibility. It might be become a promising assay for clinical diagnosis and early detection of tumors.     

AC impedance spectroscopy of native DNA and M-DNA

Monolayers of thiol-labeled DNA duplexes of 15, 20, and 30 basepairs were assembled on gold electrodes. Electron transfer was investigated by electrochemical impedance spectroscopy with Fe(CN)(6)(3-/4-) as a redox probe. The spectra, in the form of Nyquist plots, were analyzed with a modified Randles circuit which included an additional component in parallel, R(x), for the resistance through the DNA. For native B-DNA R(x) and R(ct), the charge transfer resistance, both increase with increasing length. M-DNA was formed by the addition of Zn(2+) at pH 8.6 and gave rise to characteristic changes in the Nyquist plots which were not observed upon addition of Mg(2+) or at pH 7.0. R(x) and R(ct) also increased with increasing duplex length for M-DNA but both were significantly lower compared to B-DNA. Therefore, electron transfer via the metal DNA film is faster than that of the native DNA film and certain metal ions can modulate the electrochemical properties of DNA monolayers. The results are consistent with an ion-assisted long-range polaron hopping mechanism for electron transfer.     

Construction and application of an amperometric xanthine biosensor based on zinc oxide nanoparticles-polypyrrole composite film

Zinc oxide nanoparticles (ZnO-NPs) were synthesized from zinc nitrate by simple and efficient method in aqueous media at 55°C without any requirement of calcinations step. A mixture of ZnO-NPs and pyrrole was eletropolymerized on Pt electrode to form a ZnO-NPs-polypyrrole (PPy) composite film. Xanthine oxidase (XOD) was immobilized onto this nanocomposite film through physiosorption. The ZnO-NPs/polypyrrole/Pt electrode was characterized by Fourier transform infrared (FTIR), cyclic voltammetry (CV), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical impedance spectroscopy (EIS) before and after immobilization of XOD. The XOD/ZnO-NPs-PPy/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode were connected through a potentiostat to construct a xanthine biosensor. The biosensor exhibited optimum response within 5s at pH 7.0, 35°C and linearity from 0.8 μM to 40 μM for xanthine with a detection limit 0.8 μM (S/E=3). Michaelis Menten constant (K(m)) for xanthine oxidase was 13.51 μM and I(max) 0.071 μA. The biosensor measured xanthine in fish meat and lost 40% of its initial activity after its 200 uses over 100 days, when stored at 4°C.     

Electrical impedimetric biosensors for liver function detection

In this study, electrical impedimetric biosensors composed of Au-electrodes were fabricated for the quantitative detection of human serum albumin (HSA), an essential biomarker of liver function. The Au-electrodes were fabricated via a single-step photolithography process, and can be easily integrated in biochips for assessing liver function in the future. The glass sensing surface between two adjacent Au-electrodes was modified with 3-aminopropyltriethoxysilane (APTES) to improve the biocompatibility for its subsequent binding to anti-human serum albumin (AHSA). The sensing surface without AHSA binding was blocked using skim milk powders, preventing possible non-specific bonding HSA conjugation. Biosensors were used to measure HSA concentration for liver function detection. The impedance between two adjacent Au-electrodes of the biosensors applied with various HSA concentrations was directly measured, and quantified using an electrochemical impedance spectroscopy system under AC conditions. The results of plotting both values in log scales indicated the impedance increased linearly with HSA conjugation increase. The limit of HSA detection was about 2'10(-4)mg/ml using the electrochemical impedimetric biosensor proposed in this work. This study demonstrates the feasibility of using electrochemical impedimetry as a bio-sensing mechanism to quantify human serum albumin concentration. The sensor proposed in this work also displays great potential for assessing liver function because of its simple detection mechanism, ease of biochip integration, and low cost.     

Development of tyrosinase biosensor based on quantum dots/chitosan nanocomposite for detection of phenolic compounds

A sensitive and simple amperometric biosensor for phenols was developed based on the immobilization of tyrosinase into CdS quantum dots/chitosan nanocomposite matrix. The nanocomposite film with porous nanostructure, excellent hydrophilicity and biocompatibility resulted in high enzyme loading, and the tyrosinase (Tyr) immobilized in this novel matrix retained its activity to a large extent. The CdS quantum dots/chitosan nanocomposite film was characterized by scanning electron microscopy and electrochemical impedance spectroscopy, and the parameters of the various experimental variables for the biosensor were optimized. Under the optimal conditions, the designed biosensor displayed a wide linear response to catechol over a concentration range of 1.0 × 10⁻⁹ to 2.0 × 10⁻⁵ M with a high sensitivity of 561 ± 9.7 mA M⁻¹ and a low detection limit down to 0.3 nM at a signal-to-noise ratio of 3. The CdS quantum dots/chitosan nanocomposites could provide a novel matrix for enzyme immobilization to promote the development of biosensing and biocatalysis.     

Nanocrystalline diamond impedimetric aptasensor for the label-free detection of human IgE

Like antibodies, aptamers are highly valuable as bioreceptor molecules for protein biomarkers because of their excellent selectivity, specificity and stability. The integration of aptamers with semiconducting materials offers great potential for the development of reliable aptasensors. In this paper we present an aptamer-based impedimetric biosensor using a nanocrystalline diamond (NCD) film as a working electrode for the direct and label-free detection of human immunoglobulin E (IgE). Amino (NH(2))-terminated IgE aptamers were covalently attached to carboxyl (COOH)-modified NCD surfaces using carbodiimide chemistry. Electrochemical impedance spectroscopy (EIS) was applied to measure the changes in interfacial electrical properties that arise when the aptamer-functionalized diamond surface was exposed to IgE solutions. During incubation, the formation of aptamer-IgE complexes caused a significant change in the capacitance of the double-layer, in good correspondence with the IgE concentration. The linear dynamic range of IgE detection was from 0.03 μg/mL to 42.8 μg/mL. The detection limit of the aptasensor reached physiologically relevant concentrations (0.03 μg/mL). The NCD-based aptasensor was demonstrated to be highly selective even in the presence of a large excess of IgG. In addition, the aptasensor provided reproducible signals during six regeneration cycles. The impedimetric aptasensor was successfully tested on human serum samples, which opens up the potential of using EIS for direct and label-free detection of IgE levels in blood serum.     

Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes

The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigitated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the presence of K(3)[Fe(CN)(6)]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K(3)[Fe(CN)(6)], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN)(6)(3-) ions through defects in the ODN monolayer determines the impedance of Au-ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K(3)[Fe(CN)(6)] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms.     

Chitosan/polyaniline hybrid conducting biopolymer base impedimetric immunosensor to detect Ochratoxin-A

Chitosan (CS)-polyaniline (PANI) hybrid conducting biopolymer film was obtained on indium-tin-oxide (ITO) electrode using electrochemical polymerization process. Fourier transform infrared (FT-IR) spectra of PANI-CS had showed covalent and hydrogen binding between PANI and CS molecules. Electrochemical impedance spectroscopy (EIS) measurements had showed low charge transfer resistance (R(CT)) of PANI-CS and PANI. Successive rabbit antibody (IgGs) immobilization on PANI-CS, CS and PANI matrixes surface were confirmed with FT-IR and EIS measurements. Ochratoxin-A (OTA) interaction with IgGs had increased R(CT) values and showed linear response up to 10 ng/mL OTA concentration in electrolyte. Relative change in R(CT) was higher in PANI-CS due to higher proportion of carboxylic and hydroxyl functionalities at PANI-CS matrix surfaces. The absolute sensitivity of PANI, CS, and PANI-CS were 16+/-6, 22+/-9 and 53+/-8 Omega mL/ng, respectively derived from slope of linear response up to 10 ng/mL with 1 ng/mL minimum detection limit.     

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1).     

Electrodeposition of polypyrrole-multiwalled carbon nanotube-glucose oxidase nanobiocomposite film for the detection of glucose

A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.     

Methyl parathion hydrolase based nanocomposite biosensors for highly sensitive and selective determination of methyl parathion

This article reports the fabrication of a nanocomposite biosensor for the sensitive and specific detection of methyl parathion. The nanocomposite sensing film was prepared via the formation of gold nanoparticles on silica particles, mixing with multiwall carbon nanotubes and subsequent covalent immobilization of methyl parathion hydrolase. The composite of the individual materials was finely tuned to offer the sensing film with high specific surface area and high conductivity. A significant synergistic effect of nanocomposites on the biosensor performance was observed in biosensing methyl parathion. The square wave voltammetric responses displayed well defined peaks, linearly proportional to the concentrations of methyl parathion in the range from 0.001 μg mL?1 to 5.0 μg mL?1 with a detection limit of 0.3 ng mL?1. The application of this biosensor in the analysis of spiked garlic samples was also evaluated. The proposed protocol can be used as a platform for the simple and fast construction of biosensors with good performance for the determination of enzyme-specific electroactive species.     

Electrophoretic transfer protein zymography

A mixture of commercial creatinine amidohydrolase (CA), creatine amidinohydrolase (CI), and sarcosine oxidase (SO) was coimmobilized covalently via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto carboxylated multiwalled carbon nanotube (c-MWCNT)/polyaniline (PANI) nanocomposite film electrodeposited over the surface of a platinum (Pt) electrode. A creatinine biosensor was fabricated using enzyme/c-MWCNT/PANI/Pt as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and electrochemical impedance spectroscopy (EIS). The biosensor detected creatinine levels as low as 0.1 μM, estimated at a signal-to-noise ratio of 3, within 5 s at pH 7.5 and 35 °C. The optimized biosensor showed a linear response range of 10 to 750 μM creatinine with sensitivity of 40 μA/mM/cm². The fabricated biosensor was successfully employed for determination of creatinine in human serum. The biosensor showed only 15% loss in its initial response after 180 days when stored at 4 °C.