Nanomaterial-based Electrochemical Sensors for Multiplex Medicinal Applications.

This review explores the advancements in nanomaterial-based electrochemical sensors for the multiplex detection of medicinal compounds. The growing demand for efficient and selective detection methods in the pharmaceutical field has prompted significant research into the development of electrochemical sensors employing nanomaterials. These materials, defined as functional materials with at least one dimension between 1 and 100 nanometers, encompass metal nanoparticles, polymers, carbon-based nanocomposites, and nano-bioprobes. These sensors are characterized by their enhanced sensitivity and selectivity, playing a crucial role in simultaneous detection and offering a comprehensive analysis of multiple medicinal complexes within a single sample. The review comprehensively examines the design, fabrication, and application of nanomaterial- based electrochemical sensors, focusing on their ability to achieve multiplex detection of various medicinal substances. Insights into the strategies and nanomaterials employed for enhancing sensor performance are discussed. Additionally, the review explores the challenges and future perspectives of this evolving field, highlighting the potential impact of nanomaterial-based electrochemical sensors on the advancement of medicinal detection technologies.

Toward the rapid diagnosis of sepsis: dendritic copper nanostructure functionalized diazonium salt modified screen-printed graphene electrode for IL-6 detection.

Sepsis, an infectious disease affecting millions of people's health worldwide each year, calls for urgent attention to an improvement of analytical devices. Chemiluminescence immunoassay is a typical diagnostic method utilized to assess the risk development of sepsis. However, due to its high-cost, delayed, and complicated procedure, the practical utilization is therefore undoubtedly limited, especially for point-of-care test. Herein, we fabricated for the first time an immunosensor based on dendritic copper nanostructures (CuNSs) combined with 4-aminobenzoic acid (4-AB, the diazonium salt) as antibody linker modified on a screen-printed graphene electrode for the early detection of the sepsis biomarker interleukin-6 (IL-6). The electrode fabrication is made by electrodeposition, thus eliminating the multistep of nanomaterial synthesis and time wasting. The resulting dendritic CuNSs significantly increase the effective surface area (1.2 times) and the sensor's performance. The morphology of this combination was characterized using CV, EIS, SEM, EDX, and FTIR techniques. In the detection process, the appearance of IL-6 suppresses the current response of the redox probe indicator measured by differential pulse voltammetry due to the antibody-antigen complex. The subtraction of signal (ΔI) was interpreted as IL-6 concentration. This sensor exhibited a linear range from 0.05 to 500 pg mL-1 with low detection limit of 0.02 pg mL-1, proving a possibility for early sepsis screening. In addition, the established immunosensor can successfully quantify IL-6 in human serum sample, in which the results agreed well with those achieved using the standard approach, further showing high practical applicability of this developed immunosensor.

Sequential Flow Controllable Microfluidic Device for G-Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV-2 Using a Pyrrolidinyl Peptide Nucleic Acid.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis.

Electrochemical capillary-driven microfluidic DNA sensor for HIV-1 and HCV coinfection analysis.

Electrochemical DNA sensors can be operated in either static or flow-based detection schemes. In static schemes, manual washing steps are still necessary, resulting in a tedious and time-consuming process. In contrast, in flow-based electrochemical sensors, the current response is collected when the solution flows through the electrode continuously. However, the drawback of such a flow system is the low sensitivity due to the limited time for the interaction between the capturing element and the target. Herein, we propose a novel electrochemical capillary-driven microfluidic DNA sensor to combine the advantages of static and flow-based electrochemical detection systems into a single device by incorporating burst valve technology. The microfluidic device with a two-electrode configuration was applied for the simultaneous detection of two different DNA markers, human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) cDNA, via the specific interaction between pyrrolidinyl peptide nucleic acids (PNA) probes and the DNA target. The integrated system, while requiring a small sample volume (7 µL for each sample loading port) and less analysis time, achieved good performance in terms of the limits of detection (LOD) (3SDblank/slope) and quantification (LOQ) (10SDblank/slope) at 1.45 nM and 4.79 nM for HIV and 1.20 nM and 3.96 nM for HCV, respectively. The simultaneous detection of HIV-1 and HCV cDNA prepared from human blood samples showed results that are in complete agreement with the RT‒PCR assay. The results qualify this platform as a promising alternative for the analysis of either HIV-1/HCV or coinfection that can be easily adapted for other clinically important nucleic acid-based markers.

Resistance-Based Lateral Flow Immunosensor with a NFC-Enabled Smartphone for Rapid Diagnosis of Leptospirosis in Clinical Samples.

Leptospirosis is one of the most life-threatening tropical diseases caused by pathogenic Leptospira. To date, a diagnostic device that offers rapid and sensitive detection of leptospires has been still in demand for proper treatment to reduce the mortality rate. Herein, we create a resistance-based lateral flow immunosensor diagnosis device (R-LFI) that integrates near-field communication (NFC) with a portable smartphone for leptospiral detection in clinical samples. A specific monoclonal antibody against the pathogen was coated on a nitrocellulose membrane (NCM) where the test line was collocated. Two electrodes with a sandwich-like configuration were installed employing a conductive double-sided adhesive tape and connected with a NFC smartphone-based detection system. A half-sandwich immunocomplex formation induced high proton conduction, resulting in a considerable decrement in resistive response. The performance of the R-LFI sensor was evaluated using recombinant LipL32 (rLipL32), Leptospira interrogans, and clinical samples. The R-LFI device exhibited linear responses toward rLipL32 protein in phosphate buffer and L. interrogans-spiked healthy human serum samples within the concentration ranging from 1 to 1000 ng mL-1 (limit of detection (LOD): 0.29 ng mL-1) and from 104 to 106 cell mL-1 (LOD: 4.89 × 103 cell mL-1), respectively. Our R-LFI sensor successfully detected L. interrogans-positive clinical samples as confirmed by polymerase chain reaction (PCR). This platform offers high specificity, selectivity, simplicity, miniscule sample volume, and no labeling element requirement. These desirable features make it particularly suitable for countries where medical facilities and resources are limited.

Sequential electrodeposition of Cu-Pt bimetallic nanocatalysts on boron-doped diamond electrodes for the simple and rapid detection of methanol.

In this work, a novel electrochemical sensor for methanol determination was established by developing a bimetallic catalyst with superiority to a monometallic catalyst. A Cu-Pt nanocatalyst was proposed and easily synthesized by sequential electrodeposition onto a boron-doped diamond (BDD) electrode. The successful deposition of this nanocatalyst was then verified by scanning electron microscopy and energy dispersive spectroscopy. The electrodeposition technique and sequence of metal deposition significantly affected the surface morphology and electrocatalytic properties of the Cu-Pt nanocatalyst. The presence of Cu atoms reduced the adsorption of other species on the Pt surface, consequently enhancing the long-term stability and poisoning tolerance of Pt nanocatalysts during the methanol oxidation process. This advanced sensor was also integrated with sequential injection analysis to achieve automated and high-throughput analysis. This combination can significantly improve the detection limit of the developed sensor by approximately 100 times compared with that of the cyclic voltammetric technique. The limit of detection of this sensor was 83 µM (S/N = 3), and wide linearity of the standard curve for methanol concentrations ranging from 0.1 to 1000 mM was achieved. Finally, this proposed sensor was successfully applied to detect methanol in fruit and vegetable beverage samples.

Enzyme-free impedimetric biosensor-based molecularly imprinted polymer for selective determination of L-hydroxyproline.

This study first reported enzyme-free impedimetric biosensor-based molecularly imprinted polymers for selective and sensitive determination of L-hydroxyproline (L-hyp), a biomarker for the early diagnosis of bone diseases. In recent study, utilizing a single 3-aminophenylboronic acid (3-APBA) to create imprinted surfaces could result in a strong interaction and difficulty in removal of a template molecule. Hence, a mixture of monomer solution containing 3-APBA and o-phenylenediamine (OPD) in the presence of the L-hyp molecule was co-electropolymerized onto the screen-printed electrode using cyclic voltammetry (CV) to eradicate this mentioned limitation. The detection principle of this sensor is relied on alteration of mediator's charge transfer resistance (Rct) that could be obstructed by L-hyp occupied in imprinted surface. The successfully fabricated biosensor was explored by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and confocal scanning microscopy. Furthermore, the effect of polymer composition on the Rct response was systematically investigated. The result exhibited that the mixture of monomers could provide the highest change of Rct due to high selectivity from esterification of 3-APBA and from hydrogen bond of OPD surrounding the template. The sensor showed a significant increase in Rct in the presence of L-hyp, whereas no observable resistance change was detected in the absence thereof. The calibration curve was obtained in the range from 0.4 to 25 µg mL-1 with limits of detection (3SDblank/Slope) and quantification (10SDblank/Slope) of 0.13 and 0.42 µg mL-1, respectively. This biosensor exhibited high selectivity and sensitivity and was successfully applied to determine L-hyp in human serum samples with satisfactory results.

Ultrasensitive electrochemiluminescence sensor based on nitrogen-decorated carbon dots for Listeria monocytogenes determination using a screen-printed carbon electrode.

Current method for identification of foodborne pathogens suffers from its relatively poor performance, consequently limiting its use. Herein, we first describe an ultrasensitive electrochemiluminescence (ECL) sensor based on nitrogen-decorated carbon dots (NCDs) for Listeria monocytogenes (L. monocytogenes) determination using a screen-printed carbon electrode (SPCE). Citric acid serves as carbon source, and ethylenediamine, a molecule containing nitrogen atom, is employed to synthesize CDs. Approximately 4 nm NCD with homogenous size distribution can be produced via a single step green microwave-assisted methodology. The construction of ECL sensor is initiated by the immobilization of capture antibody (Ab1) onto the carboxyl graphene (GOOH)-modified SPCE, where immunocomplexes (antigen and the NCD-labelled secondary antibody (Ab2-NCD)) are formed, resulting in a substantial increment in the ECL signal response in the presence of K2S2O8. The GOOH allows direct formation of the capture antibodies and enhances the electrochemical properties. Under optimal parameters, this sensor exhibits wide linearity (2 to 1.0 × 106 CFU mL-1), high sensitivity (0.104 or 1.0 × 10-1 CFU mL-1) and specificity over the nontargeting studied pathogens and is successfully applied to determine L. monocytogenes in food products. These promising results together with its performance suggest that this proposed platform may serve as an alternative device to effectively control the spread of foodborne diseases.

Cost-effective paper-based electrochemical immunosensor using a label-free assay for sensitive detection of ferritin.

Ferritin, a blood cell protein containing iron, is a crucial biomarker that is used to estimate the risk assessment of iron deficiency anemia. For point-of-care analysis, a reliable, cost-effective, selective, sensitive, and portable tool is extremely necessary. In this study, a label-free electrochemical immunosensor for detecting ferritin using a paper-based analytical device (ePAD) was created. The device pattern was custom designed onto filter paper to successfully fabricate a deliverable immunosensor. Graphene oxide was first modified onto the working electrode using an inkjet printing technique. An activation step of the electrode surface was then performed using standard 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysulfosuccinimide (sulfo-NHS) chemistry. Anti-ferritin antibodies were covalently immobilized onto the amine-reactive ester surface. The amount of ferritin was monitored by observing the electrochemical signal of the selected redox couple by differential pulse voltammetry (DPV). In the presence of ferritin, the sensor showed a considerable decrease in electrochemical response in a concentration-dependent manner. In contrast, there was no observable change in current response detected in the absence of ferritin. The current response provided a good correlation with ferritin concentrations in the range of 1 to 1000 ng mL-1, and the limit of detection (3SD/slope) was found to be 0.19 ng mL-1. This fabricated immunosensor offered good selectivity, reproducibility, and long-term storage stability. In addition, this proposed immunosensor was successfully applied to detect ferritin in human serum with satisfactory results. The promising results suggested that this handmade paper-based immunosensor may be an alternative device for the diagnosis of iron deficiency anemia.

Wide electrochemical window of screen-printed electrode for determination of rapamycin using ionic liquid/graphene composites.

A disposable screen-printed carbon electrode (SPCE) modified with an ionic liquid/graphene composite (IL/G) exhibits a wider potential window, excellent conductivity, and specific surface area for the improvement in the voltammetric signal of rapamycin detection. The modified composite was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). The electrochemical behavior of rapamycin at the modified SPCE was investigated by cyclic and square wave voltammetry in 60:40 EtOH: 0.1 M LiClO4 at pH 5.0. A high reproducible and well-defined peak with a high peak current were obtained for rapamycin detection at a position potential of + 0.98 V versus Ag/AgCl. Under the optimized conditions, the rapamycin concentration in the range 0.1 to 100 µM (R2 = 0.9986) had a good linear relation with the peak current. The detection limit of this method was 0.03 µM (3SD/slope). The proposed device can selectively detect rapamycin in the presence of commonly interfering compounds. Finally, the proposed method was successfully applied to determine rapamycin in urine and blood samples with excellent recoveries. These devices are disposable and cost-effective and might be used as an alternative tool for detecting rapamycin in biological samples and other biological compounds. Graphical abstract Schematic presentation of wide electrochemical window and disposable screen-printed sensor using ionic liquid/graphene composite for the determination of rapamycin. This composite can enhance the oxidation current and expand the potential for rapamycin detection.

Electrochemical immunosensor based on gold-labeled monoclonal anti-LipL32 for leptospirosis diagnosis.

Leptospirosis is a critical human health problem in the tropical area, thus, a precise technique that can be used for point-of-care analysis is greatly required. This is the first report on electrochemical immunosensor based on gold-labeled monoclonal anti-LipL32 for rapid, simple and sensitive determination of LipL32. The sensor consisted of two LipL32-specific antibodies: an unlabeled capture primary antibody (Anti-1°Ab) and an electrochemically detectable gold-conjugated secondary antibody (Au-2°Ab). The Anti-1°Ab was immobilized onto the modified screen-printed graphene electrode (SPGE) to form the anti-LipL32 surface. The electrochemical signal response was determined by differential pulse voltammetry (DPV). In the presence of LipL32, the sensor displayed a significant increase in current response in a concentration-dependent manner, but no observable signal was detected in the absence of LipL32. The linearity between LipL32 concentration and the measured current was found in a range of 1-100 ng/mL, and the limit of detection (LOD) (3SDblank/Slope) and limit of quantitation (LOQ) (10SDblank/Slope) were found to be 0.28 and 0.93 ng/mL, respectively. This sensor was successfully applied to detect pathogenic Leptospira whole cell lysates samples with the satisfactory results. The promissing results suggested that this immunosensor might be an alternative tool for diagnosis of leptospirosis.

An origami paper-based electrochemical immunoassay for the C-reactive protein using a screen-printed carbon electrode modified with graphene and gold nanoparticles.

An origami paper-based electrochemical immunoassay for C-reactive protein (CRP) detection is described. The assay integrates multiple steps of electrode modification into a single device. A graphene-modified screen-printed carbon electrode (G/SPCE) was employed to enhance sensitivity. Gold nanoparticles were first electrodeposited onto the G/SPCE, followed by a self-assembled monolayer of L-cysteine. The capture anti-CRP was then covalently immobilized on the modified electrode. CRP was quantified by measuring the changes in the charge-transfer resistance of the electrode by using hexacyanoferrate as the redox probe. Cyclic voltammetry and scanning electron microscopy were also applied to verify the successful modification of the electrode. Under optimal conditions, impedance increase in the 0.05-100 µg mL-1 CRP concentration range, and the limit of detection is 15 ng mL-1 (at S/N = 3). The immunoassay was successfully applied to the determination of CRP in a certified human serum sample. This method is simple, low-cost, portable and disposable. Graphical abstract An origami paper-based analytical device (oPAD) is described that integrates the multistep of electrode modification, immobilization and detection into a single device. The direct conjugation between the capture antibody and target molecule was allowed to use in this system. The C-reactive protein (CRP) concentration in serum samples was determined using electrochemical impedance spectroscopy.

Electrochemical detection of c-reactive protein based on anthraquinone-labeled antibody using a screen-printed graphene electrode.

In this present work, a novel electrochemical immunosensor employing a screen-printed graphene electrode (SPGE) for a simple and highly sensitive determination of C-reactive protein (CRP) in a sandwich-type format was proposed. The sensor comprised of two CRP-specific antibodies: an unlabeled capture primary antibody (Anti-1°Ab) and an electrochemically detectable anthraquinone-labeled signaling secondary (AQ-2°Ab) antibody. The Anti-1°Ab was first covalently anchored onto an L-cysteine/gold-modified disposable SPGE (L-Cys/Au/SPGE) to create the anti-CRP surface. After binding of the CRP and the AQ-2°Ab, the electrochemical signal response was measured using differential pulse voltammetry (DPV). In the presence of CRP, the sensor exhibited a significant increase in the AQ current at AQ-2°Ab compared to the negative control. The CRP concentration was detected in the range of 0.01-150 µg/mL, and the limit of detection (LOD) (S/N = 3) and limit of quantitation (LOQ) (10 SD/Slope) were 1.5 ng/mL and 10 ng/mL, respectively. This sensor exhibited very high sensitivity in determining CRP and was successfully applied to detect CRP in certified human serum with satisfactory results. The developed sensor is suitable as an alternative method for determination of CRP and the same principle may be further applied to determine other clinically important target molecules.

Electrochemically reduced graphene oxide-modified screen-printed carbon electrodes for a simple and highly sensitive electrochemical detection of synthetic colorants in beverages.

A simple and highly sensitive electrochemical sensor based on an electrochemically reduced graphene oxide-modified screen-printed carbon electrode (ERGO-SPCE) for the simultaneous determination of sunset yellow (SY) and tartrazine (TZ) was proposed. An ERGO film was coated onto the electrode surface using a cyclic voltammetric method and then characterized by scanning electron microscopy (SEM). In 0.1M phosphate buffer at a pH of 6, the two oxidation peaks of SY and TZ appeared separately at 0.41 and 0.70V, respectively. Surprisingly, the electrochemical response remarkably increased approximately 90- and 20-fold for SY and TZ, respectively, using the modified electrode in comparison to the unmodified electrode. The calibration curves exhibited linear ranges from 0.01 to 20.0µM for SY and from 0.02 to 20.0µM for TZ. The limits of detection were found to be 0.50 and 4.50nM (at S/N=3) for SY and TZ, respectively. Furthermore, this detection platform provided very high selectivity for the measurement of both colorants. This electrochemical sensor was successfully applied to determine the amount of SY and TZ in commercial beverages. Comparison of the results obtained from this proposed method to those obtained by an in-house standard technique proved that this developed method has good agreement in terms of accuracy for practical applications. This sensor offers an inexpensive, rapid and sensitive determination. The proposed system is therefore suitable for routine analysis and should be an alternative method for the analysis of food colorants.

Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 µL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

Electrochemical detection of human papillomavirus DNA type 16 using a pyrrolidinyl peptide nucleic acid probe immobilized on screen-printed carbon electrodes.

An electrochemical biosensor based on an immobilized anthraquinone-labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probe was successfully developed for the selective detection of human papillomavirus (HPV) type 16 DNA. A 14-mer acpcPNA capture probe was designed to recognize a specific 14 nucleotide region of HPV type 16 L1 gene. The redox-active label anthraquinone (AQ) was covalently attached to the N-terminus of the acpcPNA probe through an amide bond. The probe was immobilized onto a chitosan-modified disposable screen-printed carbon electrode via a C-terminal lysine residue using glutaraldehyde as a cross-linking agent. Hybridization with the target DNA was studied by measuring the electrochemical signal response of the AQ label using square-wave voltammetric analysis. The calibration curve exhibited a linear range between 0.02 and 12.0 µM with a limit of detection and limit of quantitation of 4 and 14 nM, respectively. This DNA sensing platform was successfully applied to detect the HPV type 16 DNA from a PCR amplified (240 bp fragment of the L1 gene) sample derived from the HPV type 16 positive human cancer cell line (SiHa), and failed to detect the HPV-negative c33a cell line. The sensor probe exhibited very high selectivity for the complementary 14 base oligonucleotide over the non-complementary oligonucleotides with sequences derived from HPV types 18, 31 and 33. The proposed sensor provides an inexpensive tool for the early stage detection of HPV type 16, which is an important biomarker for cervical cancer.