3D Simulation-Driven Design of a Microfluidic Immunosensor for Real-Time Monitoring of Sweat Biomarkers.

This study presents the design and comprehensive 3D multiphysics simulation of a novel microfluidic immunosensor for non-invasive, real-time detection of pro-inflammatory biomarkers in human sweat. The patch-like device integrates magnetofluidic manipulation of antibody-functionalized magnetic nanoparticles (MNPs) with direct-field capacitive sensing (DF-CS). This unique combination enhances sensitivity, reduces parasitic capacitance, and enables a more compact design compared to traditional fringing-field approaches. A comprehensive 3D multiphysics simulation of the device, performed using COMSOL Multiphysics, demonstrates its operating principle by analyzing the sensor's response to changes in the dielectric properties of the medium due to the presence of magnetic nanoparticles. The simulation reveals a sensitivity of 42.48% at 85% MNP occupancy within the detection zone, highlighting the sensor's ability to detect variations in MNP concentration, and thus indirectly infer biomarker levels, with high precision. This innovative integration of magnetofluidic manipulation and DF-CS offers a promising new paradigm for continuous, non-invasive health monitoring, with potential applications in point-of-care diagnostics, personalized medicine, and preventive healthcare.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection.

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 µg kg-1 and 10 µg kg-1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.

A microfluidic immunosensor based on magnetic separation for rapid detection of okadaic acid in marine shellfish.

Okadaic acid (OA) is a marine biotoxin that accumulates in seafood and can cause diarrheic shellfish poisoning if consumed. Accordingly, many countries have established regulatory limits for the content of OA in shellfish. At present, methods used for the detection of marine toxins are time-consuming and labor-intensive. In order to realize rapid, simple, and accurate detection of OA, we developed a novel microfluidic immunosensor based on magnetic beads modified with a highly specific and sensitive monoclonal antibody (mAb) against OA that is used in conjunction with smartphone imaging to realize the rapid detection of OA in shellfish. The method achieves on-site detection results within 1 h with an IC50 value of 3.30 ng/mL for OA and a limit of detection (LOD) of 0.49 ng/mL. In addition, the analysis of real samples showed that the recoveries for spiked shellfish samples ranged from 84.91% to 95.18%, and the results were confirmed by indirect competitive enzyme-linked immunosorbent assay (icELISA), indicating that the method has good accuracy and precision. Furthermore, the results are reported in a specially designed smartphone app. The microfluidic immunosensor has the advantages of simple operation, rapid detection, and high sensitivity, providing a reliable technical solution for detecting OA residues in shellfish.

A Novel, Quick, and Reliable Smartphone-Based Method for Serum PSA Quantification: Original Design of a Portable Microfluidic Immunosensor-Based System.

We describe a versatile, portable, and simple platform that includes a microfluidic electrochemical immunosensor for prostate-specific antigen (PSA) detection. It is based on the covalent immobilization of the anti-PSA monoclonal antibody on magnetic microbeads retained in the central channel of a microfluidic device. Image flow cytometry and scanning electron microscopy were used to characterize the magnetic microbeads. A direct sandwich immunoassay (with horseradish peroxidase-conjugated PSA antibody) served to quantify the cancer biomarker in serum samples. The enzymatic product was detected at -100 mV by amperometry on sputtered thin-film electrodes. Electrochemical reaction produced a current proportional to the PSA level, with a linear range from 10 pg mL-1 to 1500 pg mL-1. The sensitivity was demonstrated by a detection limit of 2 pg mL-1 and the reproducibility by a coefficient of variation of 6.16%. The clinical performance of this platform was tested in serum samples from patients with prostate cancer (PCa), observing high specificity and full correlation with gold standard determinations. In conclusion, this analytical platform is a promising tool for measuring PSA levels in patients with PCa, offering a high sensitivity and reduced variability. The small platform size and low cost of this quantitative methodology support its suitability for the fast and sensitive analysis of PSA and other circulating biomarkers in patients. Further research is warranted to verify these findings and explore its potential application at all healthcare levels.

Ultrasensitive microfluidic immunosensor with stir bar enrichment for point-of-care test of Staphylococcus aureus in foods triggered by DNAzyme-assisted click reaction.

This work demonstrated an ultrasensitive and simple microfluidic immunosensor for point-of-care test of Staphylococcus aureus (S. aureus) based on the stir bar enrichment and DNAzyme-assisted click reaction. Initially, S. aureus was enriched by the 4-mercaptophenylboronic acid-functionalized stir bar. The yolk antibody (immunoglobulin Y) and copper-labeled polydopamine nanoparticles were then specifically conjugated with the captured target. The Cu(II) was released under acidic conditions and effectively catalyzed the copper-catalyzed azide-alkyne cycloaddition (CuAAC) between the alkyne group-labeled DNAzyme and the streptavidin-biotin-azido with the assistance of DNAzyme. Finally, the DNAzyme-streptavidin complexes were detected by microfluidic chips to quantify S. aureus. Under optimum conditions, this immunosensor showed good detection performances toward S. aureus within 10 to 2.5 × 104 CFU/mL with a limit of detection of 3 CFU/mL. Moreover, the satisfying detection results of real samples of animal origin also implied that this immunosensor owned great potential in practical applications.

Surface-modified mesoporous nanofibers for microfluidic immunosensor with an ultra-sensitivity and high signal-to-noise ratio.

How to balance the sensitivity and signal-to-noise ratio of immunosensor remains many challenges during various diseases diagnosis. Here we develop a new microfluidic immunosensor based on surface-modified mesoporous nanofibers, and simultaneously realize an ultra-sensitivity and high signal-to-noise ratio for the detection of multiple biomarkers. In the current study, we fabricated titanium dioxide (TiO2)-based mesoporous electrospinning nanofibers, and modified nanofiber surface with both octadecylphosphonic acid (OPA) and poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (PEO-PPO-PEO). Such nanofibers as solid substrate are covered on microfluidic channels. The porosity of our nanofibers dramatically increased the adsorption capability of antibodies, realizing an ultra sensitivity of biomarker detection. PEO-PPO-PEO modification can significantly block non-specific absorptions, obtaining a satisfied signal-to-noise ratio. For the detection of HIV p24 and interleukin 5 (IL-5), our immunosensor increased 6.41 and 6.93 fold in sensitivity and improved 504.66% and 512.80% in signal-to-noise ratio, in compared with gold standard immunoassay (ELISA) used in the clinic. Our immunosensor also broaden the linear range for the detection of HIV p24 (0.86-800 pg/ml) and IL-5 (0.70-800 pg/ml), in compared with ELISA which is 5.54-500 pg/ml for HIV p24 and 4.84-500 pg/ml for IL-5. Our work provided a guideline for the construction of advanced point-of-care immunosensor with an ultra-sensitivity and high signal-to-noise ratio for disease diagnosis.

Electrochemical microfluidic immunosensor based on TES-AuNPs@Fe3O4 and CMK-8 for IgG anti-Toxocara canis determination.

We report a microfluidic immunosensor for the electrochemical determination of IgG antibodies anti-Toxocara canis (IgG anti-T. canis). In order to improve the selectivity and sensitivity of the sensor, core-shell gold-ferric oxide nanoparticles (AuNPs@Fe3O4), and ordered mesoporous carbon (CMK-8) in chitosan (CH) were used. IgG anti-T. canis antibodies detection was carried out using a non-competitive immunoassay, in which excretory secretory antigens from T. canis second-stage larvae (TES) were covalently immobilized on AuNPs@Fe3O4. CMK-8-CH and AuNPs@Fe3O4 were characterized by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry, cyclic voltammetry, electrochemical impedance spectroscopy, and N2 adsorption-desorption isotherms. Antibodies present in serum samples immunologically reacted with TES, and then were quantified by using a second antibody labeled with horseradish peroxidase (HRP-anti-IgG). HRP catalyzes the reduction from H2O2 to H2O with the subsequent oxidation of catechol (H2Q) to p-benzoquinone (Q). The enzymatic product was detected electrochemically at _100 mV on a modified sputtered gold electrode. The detection limit was 0.10 ng mL-1, and the coefficients of intra- and inter-assay variation were less than 6%, with a total assay time of 20 min. As can be seen, the electrochemical immunosensor is a useful tool for in situ IgG antibodies anti-T. canis determination.

EGFR detection in extracellular vesicles of breast cancer patients through immunosensor based on silica-chitosan nanoplatform.

In the present work, we designed a microfluidic electrochemical immunosensor with enough sensibility and precision to quantify epithermal growth factor receptor (EGFR) in plasma extracellular vesicles (EVs) of plasma from breast cancer patients. The sensor employs SiNPs coated with chitosan (SiNPs-CH) as reaction's platform, based on the covalently immobilization of monoclonal anti-EGFR on SiNPs-CH retained in the central channel (CC) of the microfluidic device. The synthetized SiNPs-CH were characterized by UV-visible spectroscopy (UV-visible), energy dispersive spectrometry (EDS), Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). EGFR was quantified by a direct sandwich immunoassay measuring through a horseradish peroxidase (HRP)-conjugated anti-EGFR. The enzymatic product (benzoquinone) was detected by reduction at - 100 mV on a sputtering gold electrode. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0 ng mL-1 to 50 ng mL-1. The detection limit was 1.37 pg mL-1, and the within- and between-assay coefficients of variation were below 6.25%. Finally, plasma samples from 30 early breast cancer patients and 20 healthy donor were analyzed by the novel method. EGFR levels in EVs (EVs-EGFR) were significantly higher than in the healthy control group (p = 0.002) and also, more sensitivity and specificity than normal serum markers like CEA and CA15.3 has been observed. EVs-EGFR concentration correlates with EGFR tumor status (p = 0.0003) as well as it correlate with the tumor size and pathological grade. To conclude, plasma EVs are suitable for proteomic characterization of cancer disease, as long as the employed method has sufficient sensitivity, like the case of immune-electrochemical nanosensors with incremented reaction surface.

Development of a nanostructured immunosensor for early and in situ detection of Xanthomonas arboricola in agricultural food production.

We report a microfluidic electrochemical immunosensor for Xanthomonas arboricola (XA) determination, based on the covalently immobilization of monoclonal anti-XA antibody (anti-XA) on a previously amino functionalized SBA-15 in situ synthesized in the central channel of a glass-poly(dimethylsiloxane) microfluidic immunosensor. The synthetized amino-SBA-15 was characterized by N2 adsorption-desorption isotherm, scanning electron microscopy and infrared spectroscopy. XA was detected by a direct sandwich immunoassay through an alkaline phosphatase (AP) enzyme-labeled anti-XA conjugate. Later, the substrate p-aminophenyl phosphate was converted to p-aminophenol by AP. The enzymatic product was detected at +100mV on a sputtered gold electrode. The measured current was directly proportional to the level of XA in walnut trees samples. The linear range was from 5 × 102 to 1 × 104CFUmL-1. The detection limit was 1.5 × 102CFUmL-1, and the within- and between-assay coefficients of variation were below 5%. Microfluidic immunosensor is a very promising tool for the early and in situ diagnosis of XA in walnuts avoiding serious economic losses.

Microfluidic immunosensor based on mesoporous silica platform and CMK-3/poly-acrylamide-co-methacrylate of dihydrolipoic acid modified gold electrode for cancer biomarker detection.

We report a hybrid glass-poly (dimethylsiloxane) microfluidic immunosensor for epidermal growth factor receptor (EGFR) determination, based on the covalent immobilization of anti-EGFR antibody (anti-EGFR) on amino-functionalized mesoporous silica (AMS) retained in the central channel of a microfluidic device. The synthetized AMS was characterized by N2 adsorption-desorption isotherm, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS) and infrared spectroscopy. The cancer biomarker was quantified in human serum samples by a direct sandwich immunoassay measuring through a horseradish peroxidase-conjugated anti-EGFR. The enzymatic product was detected at -100 mV by amperometry on a sputtering gold electrode, modified with an ordered mesoporous carbon (CMK-3) in a matrix of poly-acrylamide-co-methacrylate of dihydrolipoic acid (poly(AC-co-MDHLA)) through in situ copolymerization. CMK-3/poly(AC-co-MDHLA)/gold was characterized by cyclic voltammetry, EDS and SEM. The measured current was directly proportional to the level of EGFR in human serum samples. The linear range was from 0.01 ng mL-1 to 50 ng mL-1. The detection limit was 3.03 pg mL-1, and the within- and between-assay coefficients of variation were below 5.20%. The microfluidic immunosensor is a very promising device for the diagnosis of several kinds of epithelial origin carcinomas.

Microfluidic immunosensor for rapid and highly-sensitive salivary cortisol quantification.

This paper presents a novel poly(dimethylsiloxane) (PDMS) microfluidic immunosensor that integrates a complementary metal-oxide-semiconductor (CMOS) optical detection system for a rapid and highly-sensitive quantification of salivary cortisol. The simple and non-invasive method of saliva sampling provides an interesting alternative to the blood, allowing a fast sampling at short intervals, relevant for many clinical diagnostic applications. The developed approach is based on the covalent immobilization of a coating antibody (Ab), a polyclonal anti-IgG, onto a treated PDMS surface. The coating Ab binds the capture Ab, an IgG specific for cortisol, allowing its correct orientation. Horseradish peroxidase (HRP)-labelled cortisol is added to compete with the cortisol in the sample, for the capture Ab binding sites. The HRP-labelled cortisol, bonded to the capture Ab, is measured through the HRP enzyme and the tetramethylbenzidine (TMB) substrate reaction. The cortisol quantification is performed by colorimetric detection of HRP-labelled cortisol, through optical absorption at 450nm, using a CMOS silicon photodiode as the photodetector. Under the developed optimized conditions presented here, e.g., microfluidic channels geometry, immobilization method and immunoassay conditions, the immunosensor shows a linear range of detection between 0.01-20ng/mL, a limit of detection (LOD) of 18pg/mL and an analysis time of 35min, featuring a great potential for point-of-care applications requiring continuous monitoring of the salivary cortisol levels during a circadian cycle.

Integrated bio-affinity nano-platform into a microfluidic immunosensor based on monoclonal bispecific trifunctional antibodies for the electrochemical determination of epithelial cancer biomarker.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is a biomarker that is highly overexpressed on the surface of epithelial carcinoma cells. In this study, silver nanoparticles covered with polyvinyl alcohol (AgNPs-PVA) were synthesized, characterized and used in a microfluidic immunosensor based on the use of anti-EpCAM recombinant antibodies as a trapping agent. METHODS: The concentration of trapped EpCAM is then electrochemically quantified by HRP-conjugated anti-EpCAM-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude (at a working voltage as low as -0.10V) is directly proportional to the concentration of EpCAM. RESULTS: Under optimized conditions, the detection limits for the microfluidic immunosensor and a commercial ELISA were 0.8 and 13.9pg/L, respectively. The within-assay and between-assay coefficients of variation are below 6.5% for the proposed method. The immunosensor was validated by analyzing patient samples, and a good correlation with a commercial ELISA was obtained. CONCLUSIONS: The good analytical performance is attributed to the efficient immobilization of the anti-EpCAM recombinant antibodies on the AgNPs-PVA, and its high specificity for EpCAM. This microfluidic immunosensor is intended for use in diagnosis and prognosis of epithelial cancer, to monitor the disease, and to assess therapeutic efficacy.