Dual Colorimetric/Electrochemical Detection of Salmonella typhimurium Using a Laser-Induced Graphene Integrated Lateral Flow Immunoassay Strip.

Foodborne illnesses caused by the ingestion of contaminated foods or beverages are a serious concern due to the millions of reported cases per year. It is essential to develop sensitive and rapid detection methods of foodborne pathogens to ensure food safety for producers and consumers. Unfortunately, current detection techniques still suffer from time-consuming operations and the need for highly skilled personnel. Here, we introduce a highly sensitive dual colorimetric/electrochemical detection approach for Salmonella enterica serovar typhimurium (S. typhimurium) based on a laser-induced graphene-integrated lateral flow immunoassay (LIG-LFIA) strip. The LIG electrode was fabricated by laser engraving on a polyimide tape containing a pseudo silver/silver chloride reference electrode from silver sintering and chlorination. Using double-sided tape inserted into the strip, automatic sequential reagent delivery was enabled for the dual-mode signal readout by single-sample loading. A gold-deposited gold nanoparticle strategy was first employed to simultaneously obtain a colorimetric signal for early screening and a signal turn-on electrochemical response for high-sensitivity and -quantitative analysis. A superior performance of the strip was established, characterized by a short analysis time (12 min assay +15 min sample preparation), a broad working concentration range (1 cfu/10 mL to 108 cfu/mL), and the lowest limit of detection (1 ± 0.5 cfu/10 mL; mean ± standard deviation, n = 3) among reported multimode S. typhimurium detection schemes. The strip was successfully applied in the analysis of various food products without any bacterial enrichment or amplification required, and the results were comparable to those of the standard culture method.

Large-scale fabrication of ion-selective electrodes for simultaneous detection of Na+, K+, and Ca2+ in biofluids using a smartphone-based potentiometric sensing platform.

A significant bottleneck exists for mass-production of ion-selective electrodes despite recent developments in manufacturing technologies. Here, we present a fully-automated system for large-scale production of ISEs. Three materials, including polyvinyl chloride, polyethylene terephthalate and polyimide, were used as substrates for fabricating ion-selective electrodes (ISEs) using stencil printing, screen-printing and laser engraving, respectively. We compared sensitivities of the ISEs to determine the best material for the fabrication process of the ISEs. The electrode surfaces were modified with various carbon nanomaterials including multi-walled carbon nanotubes, graphene, carbon black, and their mixed suspensions as the intermediate layer to enhance sensitivities of the electrodes. An automated 3D-printed robot was used for the drop-cast procedure during ISE fabrication to eliminate manual steps. The sensor array was optimized, and the detection limits were 10-5 M, 10-5 M and 10-4 M for detection of K+, Na+ and Ca2+ ions, respectively. The sensor array integrated with a portable wireless potentiometer was used to detect K+, Na+ and Ca2+ in real urine and simulated sweat samples and results obtained were in agreement with ICP-OES with good recoveries. The developed sensing platform offers low-cost detection of electrolytes for point-of-care applications.

Portable smartphone integrated 3D-Printed electrochemical sensor for nonenzymatic determination of creatinine in human urine.

3D printing technologies are an attractive for fabricating electrochemical sensors due to their ease of operation, freedom of design, fast prototyping, low waste, and low cost. We report the fabrication of a simple 3D-printed electrochemical sensing device for non-enzymatic detection of creatinine, an important indicator of renal function. To create the 3D-printed electrodes (3DE), carbon black/polylactic acid (CB/PLA) composite filament was used. The 3DE was activated using 0.5 M NaOH via amperometry prior to use to improve electrochemical performance. To give selectivity for creatinine, the activated 3DE was modified with a copper oxide nanoparticle-ionic liquid/reduced graphene oxide (CuO-IL/rGO) composite. The modified 3DE was characterized using microscopy and electrochemistry. Cyclic voltammetry and amperometry were used to evaluate sensor performance. The modified 3DE provided electrocatalytic activity towards creatinine without enzymes. Under optimal conditions, the modified 3DE directly coupled with a portable smartphone potentiostat exhibited the linear detection range of 0.5-35.0 mM, and the limit of detection was 37.3 µM, which is sufficient for detecting creatinine in human urine samples. Furthermore, the other physiological compounds present in human urine were not detected on the modified 3DE. Therefore, the modified 3DE could be a tool for effective creatinine screening in the urine.

Label free electrochemical DNA biosensor for COVID-19 diagnosis.

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

Microfluidic Paper-Based Device for Medicinal Diagnosis.

BACKGROUND: The demand for point-of-care testing (POCT) devices has rapidly grown since they offer immediate test results with ease of use, makingthem suitable for home self-testing patients and caretakers. However, the POCT development has faced the challenges of increased cost and limited resources. Therefore, the paper substrate as a low-cost material has been employed to develop a cost-effective POCT device, known as "Microfluidic paper-based analytical devices (µPADs)". This device is gaining attention as a promising tool for medicinal diagnostic applications owing to its unique features of simple fabrication, low cost, enabling manipulation flow (capillarydriven flow), the ability to store reagents, and accommodating multistep assay requirements. OBJECTIVE: This review comprehensively examines the fabrication methods and device designs (2D/3D configuration) and their advantages and disadvantages, focusing on updated µPADs applications for motif identification. METHODS: The evolution of paper-based devices, starting from the traditional devices of dipstick and lateral flow assay (LFA) with µPADs, has been described. Patterned structure fabrication of each technique has been compared among the equipment used, benefits, and drawbacks. Microfluidic device designs, including 2D and 3D configurations, have been introduced as well as their modifications. Various designs of µPADs have been integrated with many powerful detection methods such as colorimetry, electrochemistry, fluorescence, chemiluminescence, electrochemiluminescence, and SER-based sensors for medicinal diagnosis applications. CONCLUSION: The µPADs potential to deal with commercialization in terms of the state-of-the-art of µPADs in medicinal diagnosis has been discussed. A great prototype, which is currently in a reallife application breakthrough, has been updated.

Improvement in sensitivity for lateral flow immunoassay of ferritin using novel device design based on gold-enhanced gold nanoparticles.

This work introduces a low-cost adhesive tape combined with a hydroxylamine/polyvinyl alcohol/polyethylene oxide (HA/PVA/PEO) blend film to fabricate novel devices for improving sensitivity of gold nanoparticle (AuNP)-based lateral flow immunoassays (LFIAs) via two platforms: (1) LFIA device with integrated gold enhancement and (2) LFIA device with two independent sample inlets. The detection of ferritin has been used for proof-of-concept. The adhesive tape inserted in the devices assists to separate two solutions independently flowing from two different inlets toward a nitrocellulose membrane. On-device gold enhancement was achieved by the enlargement of AuNPs via the catalytic reaction of KAuCl4 and HA using the HA/PVA/PEO blend film easily prepared via a solution-casting technique, which could delay the flow of HA released from the film for 180s and improve storage stability of the device. Under optimal conditions evaluated by naked eyes, the gold enhancement (LOD = 0.5 ng/mL) and double-sample inlet (LOD = 2 ng/mL) devices exhibited 20-fold and fivefold higher sensitivity respectively than a conventional device, verifying the sensitivity improvement. Furthermore, the proposed device was successfully detected ferritin in human serum samples within 10 min via naked-eye observation, exhibiting rapidity and simplicity of use, and the capability to perform on-site assays.

A non-enzymatic disposable electrochemical sensor based on surface-modified screen-printed electrode CuO-IL/rGO nanocomposite for a single-step determination of glucose in human urine and electrolyte drinks.

A non-enzymatic disposable electrochemical sensor coupled with an automated sample pretreatment paper-based device was developed to avoid an additional sample preparation step for glucose determination in human urine and electrolyte drinks. The automated sample pretreatment paper-based device was successfully fabricated by the simple coating of a strong alkaline solution on a patterned wax paper, and then attached on an electrochemical sensor. The nanocomposite of copper oxide nanoparticles, ionic liquid and reduced graphene oxide (CuO-IL/rGO) modified on the screen-printed carbon electrode (SPCE) was created and used as a non-enzymatic electrochemical glucose sensor. The presence of the CuO-IL/rGO nanocomposite on the screen-printed electrode surface was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDX), and X-ray diffraction spectroscopy (XRD). Under optimal conditions, glucose was measured by dropping 100 µL sample solution on the device and detected via chronoamperometry (CA) using a smartphone potentiostat controlled by Android app., providing a rapid current response within 20 s and linearity in a range of 0.03-7.0 mM with a limit of detection (LOD) of 0.14 µM. Furthermore, this developed device was successfully applied for determining glucose levels in human urine and electrolyte drinks, exporting satisfying results correlated with a commercial enzymatic glucose biosensor and labeled values of the commercial products. Therefore, this device could be an alternative device for a non-enzymatic glucose sensor with single-step sample loading, allowing for real-time analysis, low cost, portability, disposability, and on-field measurement.

A paper-based conductive immunosensor for the determination of Salmonella Typhimurium.

We report for the first time a highly sensitive and rapid quantitative method for the detection of Salmonella Typhimurium (S. Typhimurium) using a conductive immunosensor on a paper-based device (PAD). S. Typhimurium monoclonal antibodies (MA) were first immobilized on a paper-based device and then captured by S. Typhimurium. After an immunoreaction on the device, the polyclonal antibody-colloidal gold conjugate (PA-AuNPs) was dropped to bind with S. Typhimurium. After a complete sandwich reaction, a dark red color appeared on the paper-based device, which can be observed by the naked eye for a rapid screening test. The electrical conductivity of PA-AuNPs between the screen-printed electrodes on the paper-based device was also measured for an accurate quantitative analysis. The electrical conductivity correlated well with the concentration of S. Typhimurium, which was controlled by the amount of S. Typhimurium attached to the polyclonal antibody-colloidal gold conjugate. The device showed a linear correlation for the concentration of the S. Typhimurium in the range of 10-108 CFU mL-1 in a logarithmic plot, with an R2 value of 0.9882 and a limit of detection (LOD) as low as 10 CFU mL-1. This simple, highly sensitive, and rapid method for the S. Typhimurium detection was successfully performed within 30 min, and it can be developed into small portable measuring devices in order to facilitate preliminary screening tests.

A nanocomposite prepared from platinum particles, polyaniline and a Ti3C2 MXene for amperometric sensing of hydrogen peroxide and lactate.

A nanocomposite consisting of platinum particles, polyaniline and Ti3C2 MXene (Pt/PANI/MXene) was used to modify a screen-printed carbon electrode (SPCE) to obtain sensors for hydrogen peroxide and lactate. This nanocomposite was characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and X-ray powder diffraction (XRD) to determine the physical morphologies and the nanocomposite elements. The modified electrode exhibited the improved current response towards hydrogen peroxide (H2O2) compared with an unmodified electrode and provided a low detection limit of 1.0 µM. When lactate oxidase was immobilized on the modified electrode, the electrode responded to lactate via the H2O2 generated in the enzymatic reaction. The lactate assay was performed by amperometry at a constant potential of +0.3 V (vs. Ag/AgCl). The linear range was found to be from 0.005 to 5.0 mM with a detection limit of 5.0 µM for lactate. Ultimately, this biosensor was used for the determination of lactate in milk samples with high stability and reliability. Graphical abstractSchematic representation of a novel platinum particles/polyaniline/MXene nanocomposite (Pt/PANI/MXene) for screen-printed carbon electrode (SPCE) modification to enhance the specific surface area for immobilization of lactate oxidase (LOx) and use as enzymatic biosensor for lactate determination in milk sample.

A copper oxide-ionic liquid/reduced graphene oxide composite sensor enabled by digital dispensing: Non-enzymatic paper-based microfluidic determination of creatinine in human blood serum.

A paper-based analytical device (PAD) with an integrated composite electrode for non-enzymatic creatinine sensing was developed. The electrode was produced and optimization was efficiently accomplished using a rapid digital dispensing approach. The electrochemical sensor was fabricated using an HP D300 digital dispenser to deliver a copper oxide and ionic liquid composite onto an electrochemically reduced graphene modified screen-printed carbon electrode (CuO/IL/ERGO/SPCE) on a PAD. The modified electrode was characterized using electrochemical and microscopic techniques. Electrochemical detection of creatinine was performed on the SPCE using amperometry at a constant potential. Under optimized conditions, the paper-based sensor exhibited a linear range of 0.01-2.0 mM (R2 = 0.99) and the limit of detection was 0.22 µM (S/N = 3, IUPAC definition) for creatinine. The simple fabrication process, low cost, and clinically appropriate creatinine sensitivity make this device applicable for point-of-care use.

Gold nanoparticle core-europium(iii) chelate fluorophore-doped silica shell hybrid nanocomposites for the lateral flow immunoassay of human thyroid stimulating hormone with a dual signal readout.

Hybrid nanocomposite particles composed of a gold core coated with a europium(iii)-chelate fluorophore-doped silica shell (AuNPs@SiO2-Eu3+) have been synthesized and applied as antibody labels in lateral flow immunoassay (LFIA) devices for the determination of human thyroid stimulating hormone (hTSH). Labeling of monoclonal anti-hTSH antibodies with AuNPs@SiO2-Eu3+ nanocomposites allows for both colorimetric and fluorometric observation of assay results on LFIA devices, relying on visible light absorption due to the localized surface plasmon resonance of the Au-core and the fluorescence emission of the Eu(iii)-chelate-modified shell under UV hand lamp irradiation (365 nm), respectively. The possibility for a dual signal readout provides an attractive alternative for LFIAs: instantaneous naked eye observation of the AuNP colorimetric signal as in conventional LFIAs for hypothyroidism detection, and more sensitive fluorescence detection to assess hyperthyroidism. The limits of detection (LOD) for naked eye observation of LFIA devices are 5 µIU mL-1 and 0.1 µIU mL-1 for the colorimetric and fluorimetric detection, respectively. Using the fluorescence detection scheme in combination with a smartphone and digital color analysis, a quantitative linear relationship between the red intensity and the logarithmic concentration of hTSH was observed (R2 = 0.988) with an LOD of 0.02 µIU mL-1. Finally, LFIA devices were effectively applied for detecting hTSH in spiked diluted human serum with recovery values between 100-116%.

Fully Inkjet-Printed Paper-Based Potentiometric Ion-Sensing Devices.

A fully inkjet-printed disposable and low cost paper-based device for potentiometric Na+- or K+-ion sensing has been developed. A printed ionophore-based all-solid-state ion selective electrode on a graphene/poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (G/PEDOT:PSS) nanocomposite solid contact and a printed all-solid state reference electrode consisting of a pseudosilver/silver chloride electrode coated by a lipophilic salt-incorporating poly(vinyl chloride) membrane overprinted with potassium chloride have been combined on a microfluidically patterned paper substrate. Devices are built on standard filter paper using off-the-shelf materials. Ion sensing has been achieved within 180 s by simple addition of 20 µL of sample solution without electrode preconditioning. The limits of detection were 32 and 101 µM for Na+ and K+, respectively. The individual single-use sensing devices showed near Nernstian response of 62.5 ± 2.1 mV/decade (Na+) and 62.9 ± 1.1 mV/decade (K+) with excellent standard potential (E0) reproducibilities of 455.7 ± 5.1 mV (Na+) and 433.9 ± 2.8 mV (K+). The current work demonstrates the promising possibility of obtaining low-cost and disposable paper-based potentiometric sensing devices potentially manufacturable at large scales with industrial inkjet printing technology.

Graphene-polyaniline modified electrochemical droplet-based microfluidic sensor for high-throughput determination of 4-aminophenol.

We report herein the first development of graphene-polyaniline modified carbon paste electrode (G-PANI/CPE) coupled with droplet-based microfluidic sensor for high-throughput detection of 4-aminophenol (4-AP) in pharmaceutical paracetamol (PA) formulations. A simple T-junction microfluidic platform using an oil flow rate of 1.8 µL/min and an aqueous flow rate of 0.8 µL/min was used to produce aqueous testing microdroplets continuously. The microchannel was designed to extend the aqueous droplet to cover all 3 electrodes, allowing for electrochemical measurements in a single droplet. Parameters including flow rate, water fraction, and applied detection potential (Edet) were investigated to obtain optimal conditions. Using G-PANI/CPE significantly increased the current response for both cyclic voltammetric detections of ferri/ferrocyanide [Fe(CN)6](3-/4-) (10 times) and 4-AP (2 times), compared to an unmodified electrode. Using the optimized conditions in the droplet system, 4-AP in the presence of PA was selectively determined. The linear range of 4-AP was 50-500 µM (R(2) = 0.99), limit of detection (LOD, S/N = 3) was 15.68 µM, and limit of quantification (LOQ, S/N = 10) was 52.28 µM. Finally, the system was used to determine 4-AP spiked in commercial PA liquid samples and the amounts of 4-AP were found in good agreement with those obtained from the conventional capillary zone electrophoresis/UV-Visible spectrophotometry (CZE/UV-Vis). The proposed microfluidic device could be employed for a high-throughput screening (at least 60 samples h(-1)) of pharmaceutical purity requiring low sample and reagent consumption.

Sensitive electrochemical sensor using a graphene-polyaniline nanocomposite for simultaneous detection of Zn(II), Cd(II), and Pb(II).

This work describes the development of an electrochemical sensor for simultaneous detection of Zn(II), Cd(II), and Pb(II) using a graphene-polyaniline (G/PANI) nanocomposite electrode prepared by reverse-phase polymerization in the presence of polyvinylpyrrolidone (PVP). Two substrate materials (plastic film and filter paper) and two nanocomposite deposition methods (drop-casting and electrospraying) were investigated. Square-wave anodic stripping voltammetry currents were higher for plastic vs. paper substrates. Performance of the G/PANI nanocomposites was characterized by scanning electron microscopy (SEM) and cyclic voltammetry. The G/PANI-modified electrode exhibited high electrochemical conductivity, producing a three-fold increase in anodic peak current (vs. the unmodified electrode). The G/PANI-modified electrode also showed evidence of increased surface area under SEM. Square-wave anodic stripping voltammetry was used to measure Zn(II), Cd(II), and Pb(II) in the presence of Bi(III). A linear working range of 1-300 µg L(-1) was established between anodic current and metal ion concentration with detection limits (S/N=3) of 1.0 µg L(-1) for Zn(II), and 0.1 µg L(-1) for both Cd(II) and Pb(II). The G/PANI-modified electrode allowed selective determination of the target metals in the presence of common metal interferences including Mn(II), Cu(II), Fe(III), Fe(II), Co(III), and Ni(II). Repeat assays on the same device demonstrated good reproducibility (%RSD<11) over 10 serial runs. Finally, this system was utilized for determining Zn(II), Cd(II), and Pb(II) in human serum using the standard addition method.

Ultra-performance liquid chromatography coupled with graphene/polyaniline nanocomposite modified electrode for the determination of sulfonamide residues.

An ultra-performance liquid chromatography (UPLC) coupled with graphene/polyaniline (G/PANI)-modified screen-printed carbon electrode was developed for separation and sensitive determination of eight sulfonamides (SAs) in shrimp. Electrospraying was selected for electrode modification because it can generate the well dispersion of G/PANI nanocomposites on the electrode surface. Prior to electrochemical detection, eight SAs were completely separated within 7 min by using reversed phase UPLC (C4) with mobile phase containing 70:25:5 (v/v/v) of potassium hydrogen phosphate (pH 3):acetonitrile:ethanol. For amperometric detection, the detection potential acquired from hydrodynamic voltammetry was found to be +1.4V. Under optimal conditions, a wide linearity and low limit of detection were obtained for eight SAs in the range of 0.01-10 µg mL(-1) and 1.162-6.127 ng mL(-1), respectively. Compared to boron-doped diamond (BDD) electrode, a G/PANI-modified screen-printed carbon electrode offered higher sensitivity with lower operating cost. To determine SAs in shrimp samples, solid-phase extraction was used to clean up and preconcentrate the samples prior to UPLC separation. To validate this developed method, a highly quantitative agreement was accomplished with UPLC-UV system. Thus, this proposed system might be an alternative approach for rapid, inexpensive, and sensitive determination of SAs in shrimps.

Novel paper-based cholesterol biosensor using graphene/polyvinylpyrrolidone/polyaniline nanocomposite.

A novel nanocomposite of graphene (G), polyvinylpyrrolidone (PVP) and polyaniline (PANI) has been successfully prepared and used for the modification of paper-based biosensors via electrospraying. The droplet-like nanostructures of G/PVP/PANI-modified electrodes are obtained with an average size of 160 ± 1.02 nm. Interestingly, the presence of small amount of PVP (2 mg mL(-1)) in the nanocomposites can substantially improve the dispersibility of G and increase the electrochemical conductivity of electrodes, leading to enhanced sensitivity of the biosensor. The well-defined cyclic voltammogram of standard ferri/ferrocyanide is achieved on a G/PVP/PANI-modified electrode with a 3-fold increase in the current signal compared to an unmodified electrode. This modified electrode also exhibits excellent electrocatalytic activity towards the oxidation of hydrogen peroxide (H2O2). Furthermore, cholesterol oxidase (ChOx) is attached to G/PVP/PANI-modified electrode for the amperometric determination of cholesterol. Under optimum conditions, a linear range of 50 µM to 10mM is achieved and the limit of detection is found to be 1 µM for cholesterol. Finally, the proposed system can be applied for the determination of cholesterol in a complex biological fluid (i.e. human serum).

Graphene-loaded nanofiber-modified electrodes for the ultrasensitive determination of dopamine.

A novel and highly sensitive electrochemical system based on electrospun graphene/polyaniline/polystyrene (G/PANI/PS) nanofiber-modified screen-printed carbon electrodes has been developed for dopamine (DA) determination. A dramatic increase (9 times) in the current signal for the redox reaction of a standard, ferri/ferrocyanide [Fe(CN)6](3-/4-) couple was found when compared to an unmodified electrode. This modified electrode also exhibited favorable electron transfer kinetics and excellent electrocatalytic activity toward the oxidation of DA. When used together with square wave voltammetry (SWV), DA can be selectively determined in the presence of the common interferents (i.e. ascorbic acid and uric acid). Under optimal conditions, a very low limit of detection (0.05 nM) and limit of quantification (0.30 nM) were achieved for DA. In addition, a wide dynamic range of 0.1 nM to 100 µM was found for this electrode system. Finally, the system can be successfully applied to determine DA in complex biological environment (e.g. human serum, urine) with excellent reproducibility.

A fast and highly sensitive detection of cholesterol using polymer microfluidic devices and amperometric system.

In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100 s when using 40 mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68 U/mL of the ChOx, a separation voltage of +1.6 kV, an injection time of 20s, and a detection potential of +0.5 V. PDMS microchip capillary electrophoresis showed linearity between 38.7 µg/dL (1 µM) and 270.6 mg/dL (7 mM) for the cholesterol standard; the detection limit was determined as 38.7 ng/dL (1 nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9-101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels.