Applications of Metals and Metal Compounds in Improving the Sensitivity of Microfluidic Biosensors - A Review.

The enhancement of detection sensitivity in microfluidic sensors has been a continuously explored field. Initially, many strategies for sensitivity improvement involved introducing enzyme cascade reactions, but enzyme-based reactions posed challenges in terms of cost, stability, and storage. Therefore, there is an urgent need to explore enzyme-free cascade amplification methods, which are crucial for expanding the application range and improving detection stability. Metal or metal compound nanomaterials have gained great attention in the exploitation of microfluidic sensors due to their ease of preparation, storage, and lower cost. The unique physical properties of metallic nanomaterials, including surface plasmon resonance, surface-enhanced Raman scattering, metal-enhanced fluorescence, and surface-enhanced infrared absorption, contribute significantly to enhancing detection capabilities. The metal-based catalytic nanomaterials, exemplified by Fe3O4 nanoparticles and metal-organic frameworks, are considered viable alternatives to biological enzymes due to their excellent performance. Herein, we provide a detailed overview of the applications of metals and metal compounds in improving the sensitivity of microfluidic biosensors. This review not only highlights the current developments but also critically analyzes the challenges encountered in this field. Furthermore, it outlines potential directions for future research, contributing to the ongoing development of microfluidic biosensors with improved detection sensitivity.

MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples.

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

Ti3C2/Ni/Sm-based electrochemical glucose sensor for sweat analysis using bipolar electrochemistry.

An innovative electrochemical sensor is introduced that utilizes bipolar electrochemistry on a paper substrate for detecting glucose in sweat. The sensor employs a three-dimensional porous nanocomposite (MXene/NiSm-LDH) formed by decorating nickel-samarium nanoparticles with double-layer MXene hydroxide. These specially designed electrodes exhibit exceptional electrocatalytic activity during glucose oxidation. The glucose sensing mechanism involves enzyme-free oxidation of the analyte within the sensor cell, achieved by applying an appropriate potential. This leads to the reduction of K3Fe(CN)6 in the reporter cell, and the resulting current serves as the response signal. By optimizing various parameters, the measurement platform enables the accurate determination of sweat glucose concentrations within a linear range of 10 to 200 µM. The limit of detection (LOD) for glucose is 3.6 µM (S/N = 3), indicating a sensitive and reliable detection capability. Real samples were analysed  to validate the sensor's efficiency, and the results obtained were both promising and encouraging.

Gold-carbon dot (Au@Cd) nanoconjugates based electrochemical sensing of cholesterol and effect of nitrogen ion implantation on sensitivity.

Serum cholesterol dysregulation is associated with prognosis and diagnosis of many diseases and effective biosensor will improvise their management. A novel electrochemical biosensor was fabricated based on gelatin-Au@CD nanoconjugate films for cholesterol detection. Initially, the surface of indium titanium oxide (ITO) coated glass was modified by drop casting of gelatin-Au@CD nanoconjugates to prepare the electrodes. Electrochemical studies for detection of bioanalytes(such as urea (U), ascorbic acid (AA), oxalic acid (OA), gallic acid (GA), cholesterol (Chox), dextrose (D), l-cysteine (Cys) and citric acid (CA)) were performed using cyclic voltammetry. The presence of nanoconjugates provided an appropriate environment for enhanced electrochemical response for cholesterol. These electrodes exhibited a linear response towards the presence of cholesterol in the linear concentration range of 2-20 mM with a correlation coefficient of 0.95, and the superior sensitivity of 1.36 µA/mM/cm2. Additionally, enhanced sensitivity (2.99 µA/mM/cm2) of nitrogen ion irradiated films up to a fluence of 1016 ions/cm2 was noticed because of morphological changes in the electrode surface brought about by irradiation. Approximately 54% enhancement was found when the ion fluence was 1016 ions/cm2. The designed nanoconjugate electrode showed excellent response towards cholesterol sensing and eliminates the requirement of any enzymes making the overall process simpler, cost-effective and allows for room temperature storage.

Biocompatible gelatin/carbon dot nanocomposite based urea sensor and the effect of nitrogen ion implantation.

In this study, we have fabricated a novel platform for sensing of urea using gelatin/carbon dots nanocomposite system. The sensor electrode was created by depositing the nanocomposite gel onto thin glass plates coated with indium tin oxide (ITO) using the drop casting technique. The behavior of these electrodes was investigated against a number of bioanalytes in the concentration range of 2-20 mM by cyclic voltammetry. The system was observed to be highly selective for urea with a sensitivity of 1.65 µA/mM/cm in the experimental linear range of 2-20 mM. Furthermore, the gelatin/CD-ITO electrode were also subjected to 50 KeV N2+ ion beam irradiation with varying fluence in the range of 1012 to 1016 ions/cm2. Sensing profile of the irradiated samples for urea suggested enhancement in sensitivity to 2 µA/mM cm2, when the ion fluence was 5 × 1015 ions/cm2. This enhancement after irradiation suggests a clear dependence of detection on the fluence of the ion beam. The observed excellent sensitivity of radiation processed nanocomposite material can be used as an enzyme-free platform for urea detection. Additionally, the CDs showed fluorescence quenching on treatment with mere 50 µM urea suggesting the high sensitivity of the platform.

Silver Nanoclusters as Label Free Non-enzymatic Fast Glucose Assay with the Fluorescent Enhancement Signal.

A simple and low-cost green preparation method was used for BSA capped silver nanoclusters (BSA-Ag NCs) as turn on fluorescent probe for glucose. Non-enzymatic fast glucose detection assay with a widest concentration range was proposed which requires neither nanoclusters (NCs) modification nor complicated enzyme immobilization. The DLS analysis, HRTEM patterns, fluorescence and UV-visible measurement well supported the synthesis product. The advantages of the fabricated glucose sensor based on fluorescence increasing of probe compared to other established optical techniques was inspected and summarized as well. The glucose sensor exhibited a high sensitivity, fast response time (in seconds), satisfactory selectivity, well stability (at least two months), low detection limit (31 µmol L- 1) and a wide concentration response (three orders of magnitudes) to glucose between 0.1 and 92 mmol L- 1 as calibration plot. A theoretical model of the sensing mechanism based on the binding interaction of glucose to BSA-Ag NCs is proposed and data fitting demonstrated a good agreement between the experimental and theoretically calculated fluorescence data. The facile preparation and excellent sensing performance of BSA-Ag NCs in the real samples (plasma and juice) make sure that synthesized probe material is a promising candidate for advanced enzyme-free glucose sensing approach.

Recent Advances of Enzyme-Free Electrochemical Sensors for Flexible Electronics in the Detection of Organophosphorus Compounds: A Review.

Emerging materials integrated into high performance flexible electronics to detect environmental contaminants have received extensive attention worldwide. The accurate detection of widespread organophosphorus (OP) compounds in the environment is crucial due to their high toxicity even at low concentrations, which leads to acute health concerns. Therefore, developing rapid, highly sensitive, reliable, and facile analytical sensing techniques is necessary to monitor environmental, ecological, and food safety risks. Although enzyme-based sensors have better sensitivity, their practical usage is hindered due to their low specificity and stability. Therefore, among various detection methods of OP compounds, this review article focuses on the progress made in the development of enzyme-free electrochemical sensors as an effective nostrum. Further, the novel materials used in these sensors and their properties, synthesis methodologies, sensing strategies, analytical methods, detection limits, and stability are discussed. Finally, this article summarizes potential avenues for future prospective electrochemical sensors and the current challenges of enhancing the performance, stability, and shelf life.

Recent Progress in MOF-Based Electrochemical Sensors for Non-Enzymatic Glucose Detection.

In recent years, substantial advancements have been made in the development of enzyme-free glucose sensors utilizing pristine metal-organic frameworks (MOFs) and their combinations. This paper provides a comprehensive exploration of various MOF-based glucose sensors, encompassing monometallic MOF sensors as well as multi-metal MOF combinations. These approaches demonstrate improved glucose detection capabilities, facilitated by the augmented surface area and availability of active sites within the MOF structures. Furthermore, the paper delves into the application of MOF complexes and derivatives in enzyme-free glucose sensing. Derivatives incorporating carbon or metal components, such as carbon cloth synthesis, rGO-MOF composites, and core-shell structures incorporating noble metals, exhibit enhanced electrochemical performance. Additionally, the integration of MOFs with foams or biomolecules, such as porphyrins, enhances the electrocatalytic properties for glucose detection. Finally, this paper concludes with an outlook on the future development prospects of enzyme-free glucose MOF sensors.

Self-Supported 3D PtPdCu Nanowires Networks for Superior Glucose Electro-Oxidation Performance.

The development of non-enzymatic and highly active electrocatalysts for glucose oxidation with excellent durability for blood glucose sensors has aroused widespread concern. In this work, we report a fast, simple, and low-cost NaBH4 reduction method for preparing ultrafine ternary PtPdCu alloy nanowires (NWs) with a 3D network nanostructure. The PtPdCu NWs catalyst presents significant efficiency for glucose oxidation-reduction (GOR), reaching an oxidative peak-specific activity of 0.69 mA/cm2, 2.6 times that of the Pt/C catalyst (0.27 mA/cm2). Further reaction mechanism investigations show that the NWs have better conductivity and smaller electron transfer resistance. Density functional theory (DFT) calculations reveal that the alloying effect of PtPdCu could effectively enhance the adsorption energy of glucose and reduce the activation energy of GOR. The obtained NWs also show excellent stability over 3600 s through a chronoamperometry test. These self-supported ultrafine PtPdCu NWs with 3D networks provide a new functional material for building blood glucose sensors and direct glucose fuel cells.

A multifunctional fluorescent sensor for Ag+ and Hg2+ detection in seawater.

In order to detect Ag+ and Hg2+ in seawater, we explored a multifunctional fluorescence sensor. A multifunctional Ag+ and Hg2+ sensor was designed by using gold nanoparticles (AuNPs) as quenching agent, PicoGreen dye as fluorescent probe of base pairing double-stranded deoxyribonucleic acid (DNA), and combining the characteristics of Ag+ making C base mismatch and Hg2+ making T base mismatch. Meanwhile, the DNA logic gate was constructed by establishing logic circuit, truth table, and logic formula. The relevant performances of the sensor were investigated. The results revealed that the sensor can detect Ag+ in the range of 100 to 700 nM with R2 = 0.98129, and its detection limit is 16.88 nM (3σ/slope). The detection range of Hg2+is 100-900 nM with R2 = 0.99725, and the detection limit is 5.59 nM (3σ/slope). An AND-AND-NOR-AND molecular logic gate has been successfully designed. With the characteristics of high sensitivity, multifunction, and low cost, the recommended detection method has the potential to be applied to the detection of Ag+ and Hg2+ in seawater.

Electrochemical Nano-Roughening of Gold Microstructured Electrodes for Enhanced Sensing in Biofluids.

A key challenge for sensor miniaturization is to create electrodes with smaller footprints, while maintaining or increasing sensitivity. In this work, the electroactive surface of gold electrodes was enhanced 30-fold by wrinkling followed by chronoamperometric (CA) pulsing. Electron microscopy showed increased surface roughness in response to an increased number of CA pulses. The nanoroughened electrodes also showed excellent fouling resistance when submerged in solutions containing bovine serum albumin. The nanoroughened electrodes were used for electrochemical detection of Cu2+ in tap water and of glucose in human blood plasma. In the latter case, the nanoroughened electrodes allowed highly sensitive enzyme-free sensing of glucose, with responses comparable to those of two commercial enzyme-based sensors. We anticipate that this methodology to fabricate nanostructured electrodes can accelerate the development of simple, cost-effective, and high sensitivity electrochemical platforms.

A homogeneous enzyme-free ratiometric immunoassay for the determination of C-peptide.

In this study, a homogeneous enzyme-free ratiometric (HOMO- EF-RA) immunoassay was developed for the sensitive detection of C-peptide. In the immunoassay, there have been a miscible detection system by mixing with the fluorescent quantum dots conjugated antigen (QD-Ag conjugates) and the dylight dye conjugated antibody (DL-Ab conjugates). When connecting between Ag-QD conjugate and Ab-DL conjugate by specific recognition, the system emitted fluorescence resonant energy transfer (FRET). The target C-peptide can inhibit the connection and FRET formation between QD-Ag conjugates and DL-Ab conjugates, thus changing the dual fluorescence. By measuring the ratio dual fluorescence changes of the system, the content of C-peptide was evaluated without any enzyme used and multiple incubation and washing steps. This immunoassay realized the highly sensitive (as low as 0.12 ng mL-1), selective and rapid (as less as 6 min) detection of C-peptide. Furthermore, the simple and convenient immunoassay was applied successfully to the determination of C-peptide in real serum samples.

A label-free and signal-amplifiable assay method for colorimetric detection of carcinoembryonic antigen.

In this work, an innovative colorimetric assay method for the determination of carcinoembryonic antigen is developed with aptamer probes utilized as recognition element. DNA hybridization chain reaction is used as signal amplification technique, and peroxidase-mimicking hemin/G-quadruplex-assisted catalytic oxidation of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) is deployed as signal reporting mechanism. The detection principle was firstly verified by using gel electrophoresis analysis and absorbance measurements. After condition optimization, a detection limit was theoretically determined as 24.8 ng/ml. Furthermore, the method exhibited good selectivity and satisfactory recovery rates (92.2%-108.6%) in serum samples. Moreover, the sensing scheme is easily extended for the detection of other analytes via similar target-aptamer recognition principle. To sum up, this is an enzyme- and label-free, cost-effective yet signal-amplifiable assay scheme for the determination of tumor markers with promising simplicity and selectivity, practical utility, and potential universality.

A competitive immunoassay for detecting triazophos based on fluorescent catalytic hairpin self-assembly.

A rapid detection method is introduced for residual trace levels of triazophos in water and agricultural products using an immunoassay based on catalytic hairpin self-assembly (CHA). The gold nanoparticle (AuNPs) surface was modified with triazophos antibody and sulfhydryl bio-barcode, and an immune competition reaction system was established between triazophos and its ovalbumin-hapten (OVA-hapten). The bio-barcode served as a catalyst to continuously induce the CHA reaction to achieve the dual signal amplification. The method does not rely on the participation of enzymes, and the addition of fluorescent materials in the last step avoids interfering factors, such as a fluorescence burst. The emitted fluorescence was detected at 489/521 nm excitation/emission wavelengths. The detection range of the developed method was 0.01-50 ng/mL for triazophos, and the limit of detection (LOD) was 0.0048 ng/mL. The developed method correlates well with the results obtained by LC-MS/MS, with satisfactory recovery and sensitivity. In sum, the designed method is reliable and provides a new approach to detect pesticide residues rapidly and quantitatively.

Development of an optical immunoassay based on peroxidase-mimicking Prussian blue nanoparticles and a label-free electrochemical immunosensor for accurate and sensitive quantification of milk species adulteration.

In contrast to reported enzyme-based immunoassays, an enzyme-free immunoassay (optical and electrochemical) is presented here for the first time that can be used as point-of-need detection bioplatforms of bovine IgG as goat milk adulterant. In the first format, Prussian blue nanoparticles (PBNPs) were used as antibody catalytic labels in a competitive colorimetric microplate immunoassay. Absorbance measurement was performed photometrically at 450 nm. After in-depth optimization, excellent sensitivity was achieved (0.01% cow/goat volume ratio), which is 100 times lower than the limit allowed by the European legislation (EL) (1% v/v), thanks to the high catalytic activity of PBNPs compared with natural peroxidase. Moreover, the antibody-PBNPs bioconjugates showed excellent stability over 4 weeks (> 94% of the initial response) confirming the successful anchoring of the antibodies to the surface of the PBNPs. On the other hand, a label-free voltammetric immunoassay for the detection of bovine IgG was developed. The sensing principle was based on the hindrance of charge transfer between ferri-ferrocyanide redox couple and the screen-printed gold electrodes modified with bovine IgG antibody. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the step-by-step modification of the electrode surface. Under optimal conditions, this single-step electrochemical analysis achieved a high sensitivity of 0.1% (cow/goat) when monitoring the ferrocyanide oxidation at + 0.092 V (vs. Ag/AgCl) using differential pulse voltammetry (DPV). The selectivity of the developed immunoassays was evaluated for different species of milk of similar composition, and both immunoassays exhibited a selective response only to bovine IgG. Unlike conventional immunoassays, the developed enzyme-free immunoassays have many attractive features for the detection of milk adulteration, whether they are used in quality control laboratories for routine milk analysis (optical immunoassay) or at on-site checkpoints (electrochemical immunoassay) using wireless electrochemical detectors. The sensors provide high sensitivity (≤ 0.1%), excellent precision (RSD < 6%), low cost (no enzyme is required) and ease of operation, including handling of milk samples.

Nanoporous Cauliflower-like Pd-Loaded Functionalized Carbon Nanotubes as an Enzyme-Free Electrocatalyst for Glucose Sensing at Neutral pH: Mechanism Study.

In this work, we propose a novel functionalized carbon nanotube (f-CNT) supporting nanoporous cauliflower-like Pd nanostructures (PdNS) as an enzyme-free interface for glucose electrooxidation reaction (GOR) in a neutral medium (pH 7.4). The novelty resides in preparing the PdNS/f-CNT biomimetic nanocatalyst using a cost-effective and straightforward method, which consists of drop-casting well-dispersed f-CNTs over the Screen-printed carbon electrode (SPCE) surface, followed by the electrodeposition of PdNS. Several parameters affecting the morphology, structure, and catalytic properties toward the GOR of the PdNS catalyst, such as the PdCl2 precursor concentration and electrodeposition conditions, were investigated during this work. The electrochemical behavior of the PdNS/f-CNT/SPCE toward GOR was investigated through Cyclic Voltammetry (CV), Linear Sweep Voltammetry (LSV), and amperometry. There was also a good correlation between the morphology, structure, and electrocatalytic activity of the PdNS electrocatalyst. Furthermore, the LSV response and potential-pH diagram for the palladium-water system have enabled the proposal for a mechanism of this GOR. The proposed mechanism would be beneficial, as the basis, to achieve the highest catalytic activity by selecting the suitable potential range. Under the optimal conditions, the PdNS/f-CNT/SPCE-based biomimetic sensor presented a wide linear range (1-41 mM) with a sensitivity of 9.3 µA cm-2 mM-1 and a detection limit of 95 µM (S/N = 3) toward glucose at a detection potential of +300 mV vs. a saturated calomel electrode. Furthermore, because of the fascinating features such as fast response, low cost, reusability, and poison-free characteristics, the as-proposed electrocatalyst could be of great interest in both detection systems (glucose sensors) and direct glucose fuel cells.

Ginkgo Leaf Inspired Fabrication of Micro/Nanostructures and Demonstration of Flexible Enzyme-Free Glucose Sensors.

Flexible enzyme-free glucose sensors have attracted widespread attention due to their importance and potential applications in clinical diagnosis, flexible wearable devices, and implanted devices in vivo. At present, there are still major problems in fabricating flexible enzyme-free glucose sensors with low detection limits, high stability, and high sensitivity at low cost, hindering their practical application. Here, we report a facile strategy for the fabrication of flexible non-enzymatic glucose sensors using ginkgo leaf as a template. NiO film and PEDOT:PSS composite film were deposited on the surface of the ginkgo leaf induced micro-nano hierarchical structure as a sensitive layer and a conductive layer, respectively. The as-prepared, flexible, enzyme-free glucose sensor exhibited excellent electrochemical performance toward glucose oxidation with a sensitivity of 0.7413 mA·mM-1/cm-2, an operating voltage of 0.55 V, a detection limit of 0.329 µM, and good anti-interference. Due to the simple fabrication process and performance reliability, the novel flexible enzyme-free glucose sensor is an attractive candidate for next generation wearable and implantable non-enzymatic glucose diagnostic devices.

[Research Progress on Enzyme-free Blood Glucose Sensor].

Blood glucose monitoring is of great significance to diabetic patients, and the development of rapid, accurate and real-time glucose detection technology has become a research hotspot nowadays. This study introduces the concept and classification of the enzyme-free glucose sensor, expounds enzymefree glucose sensor electrode characterization methods and the application progress of different materials in enzyme-free blood glucose sensors. Meanwhile, some problems of enzyme-free glucose sensor existing in the current research and its future application prospects also will be discussed.

Multifunctional Thermoresponsive Microcarriers for High-Throughput Cell Culture and Enzyme-Free Cell Harvesting.

An effective treatment of human diseases using regenerative medicine and cell therapy approaches requires a large number of cells. Cultivation of cells on microcarriers is a promising approach due to the high surface-to-volume ratios that these microcarriers offer. Here, multifunctional temperature-responsive microcarriers (cytoGel) made of an interpenetrating hydrogel network composed of poly(N-isopropylacrylamide) (PNIPAM), poly(ethylene glycol) diacrylate (PEGDA), and gelatin methacryloyl (GelMA) are developed. A flow-focusing microfluidic chip is used to produce microcarriers with diameters in the range of 100-300 µm and uniform size distribution (polydispersity index of ≈0.08). The mechanical properties and cells adhesion properties of cytoGel are adjusted by changing the composition hydrogel composition. Notably, GelMA regulates the temperature response and enhances microcarrier stiffness. Human-derived glioma cells (U87) are grown on cytoGel in static and dynamic culture conditions with cell viabilities greater than 90%. Enzyme-free cell detachment is achieved at room temperature with up to 70% detachment efficiency. Controlled release of bioactive molecules from cytoGel is accomplished for over a week to showcase the potential use of microcarriers for localized delivery of growth factors to cell surfaces. These microcarriers hold great promise for the efficient expansion of cells for the industrial-scale culture of therapeutic cells.

Study on performance of mimic uricase and its application in enzyme-free analysis.

Nanozymes were the novel research field to replace natural enzymes because of stability and low cost. However, the research on nanozymes was mainly focused on peroxidase, and there was little research about nanozymes with oxidase-like activity, especially mimic oxidase of small molecules related to human physiology. High levels of uric acid (UA) in the body can cause hyperuricemia and gout. And natural uricase cured this disease because it could oxidize UA. The oxidase-like activity of mixed valence state metal organic frameworks with cerium (MVSM) had been studied, but MVSM was found to have uricase-like activity in this article. The catalytic process of UA with MVSM was studied by a variety of analytical methods, which was similar to the natural uricase except for further oxidation of H2O2. The catalytic activity constants of MVSM were acquired by the Michaelis-Menten equation. MVSM had a better ability to catalyze UA in in vivo and in vitro experiments. An enzyme-free analysis-based mimic uricase for UA was established. All the experimental results proved that MVSM had a good prospect to replace the natural uricase. A nanomaterial, mixed valence state Ce-MOF (MVSM), with uricase-like activity has been found in vivo and in vitro. This material has potential to be a fluorescent analysis for detecting uric acid without uricase.