Rapid and Nondestructive Evaluation of Platelet Function in Whole Blood by Microfluidic Deterministic Cytometry.

Platelet size is a determinant of platelet function. Here, a new microfluidic deterministic cytometry packed with S-shaped micropillars (S-MDC) was developed to rapidly and sensitively determine the apparent size (Dapp) of platelets, which was used to evaluate platelet function. The platelet Dapp in the diluted whole blood was rapidly and label-freely measured by S-MDC within 2 min under shear rates (0.4 mm/s) that mimicked physiological conditions. The level of CD62p on platelets scarcely changed before and after platelets went through the whole S-MDC, indicating that the platelet function was nondestructive. Notably, the human platelet Dapp determined before and after thrombin addition by S-MDC was highly coincident with the levels of CD62p on the platelet surface by flow cytometry (r = 0.819), revealing that the human platelet Dapp was available to assess the platelet activation state. In addition, the results of the rat platelet Dapp were consistent with myocardial injury of rats with myocardial ischemia before and after treatment with antiplatelet agents, suggesting that rat platelet Dapp can be used to reflect myocardial injury in vivo outcomes. These findings reveal that S-MDC is a promising technique for screening tests for a bleeding disorder, in addition to monitoring antiplatelet drugs.

PCR-Free, Label-Free, and Centrifugation-Free Diagnosis of Multiplex Antibiotic Resistance Genes by Combining mDNA-Au@Fe3O4 from Heating Dry and DNA Concatamers with G-Triplex.

Accurate identification of antibiotic resistance genes (ARGs) is crucial for improving treatment and controlling the spread of antibiotic-resistant bacteria (ARB). Herein, a novel PCR-free, centrifugation-free, and label-free magnetic fluorescent biosensor (MFB) was developed by combining polyA-medium DNA-polyT (mDNA, which contained a partial sequence of a target DNA), gold nanoparticle (AuNP)-anchored magnetic nanoparticle (Au@Fe3O4), complementary strand DNA (CS) of the target DNA, DNA concatamer with G-triplex (G3), and thioflavin T (ThT). Thereinto, Au@Fe3O4 nanoparticles were first capped by mDNA strands within 20 min using a simple hot drying method, and then CS was added and hybridized with mDNA on Au@Fe3O4. Second, a DNA concatamer was used to bind with CS on Au@Fe3O4. When an ARG was present in the sample, the CS would recognize it and release the DNA concatamer into solution by a toehold-mediated strand displacement reaction. Finally, under magnetic separation, the free DNA concatamers with G3 were taken out easily and bound with ThT, resulting in strong fluorescence signals. The fluorescence intensity of ThT was positively correlated with the concentration of the ARG. The whole analysis was accomplished within 1.5 h using 96-well plates. Remarkably, our MFB was universal; eight ARGs were detected by replacing the corresponding mDNA and CS in this study. To verify the practicability of our method, 12 clinically isolated strains were analyzed. The results of the MFB method were in good agreement with those of the quantitative real-time PCR method with an area under the curve of 0.92 (95% confidence interval: 0.8479 to 0.9932), sensitivity of 92.00%, and specificity of 91.55%. Above all, the MFB assay established here is simple, low-cost, and universal and has great potential for applications in the identification of ARGs.

High-performance cation electrokinetic concentrator based on a γ-CD/QCS/PVA composite and microchip for evaluating the activity of P-glycoprotein without any interference from serum albumin.

The development of cation electrokinetic concentrators (CECs) has been hindered by the lack of commercial anion-exchange membranes (AEMs). This paper introduces a γ-cyclodextrin-modified quaternized chitosan/polyvinyl alcohol (γ-CD/QCS/PVA) composite as an AEM, which is combined with a microchip to fabricate a CEC. Remarkably, the CEC only concentrates cationic species, thereby overcoming the interference of the highly abundant, negatively charged serum albumin in the blood sample. P-Glycoprotein (P-gp) is recognized as an efflux transporter protein that influences the pharmacokinetics (PK) of various compounds. The CEC was used to evaluate the activity of P-gp by detecting the positively charged rhodamine 123 (Rho123 is a classical substrate of P-gp) with no interference from serum albumin in the serum sample. Using the CEC, the enrichment factor (EF) of Rho123 exceeded 105-fold under DC voltage application. The minimal sample consumption of the CEC (<10 µL) enables reduction of animal sacrifice in animal experiments. Here, the CEC has been applied to evaluate the transport activity of P-gp in in vitro and in vivo experiments by detecting Rho123 in the presence of P-gp inhibitors or agonists. The results are in good agreement with those reported in previous reports. Therefore, the CEC presents a promising application potential, owing to its simple fabrication process, high sensitivity, minimal sample consumption, lack of interference from serum albumin and low cost.

Low-Cost Full-Range Detection of C-Reactive Protein in Clinical Samples by Aptamer Hairpin Probes and Coprecipitation of Silver Ions and Gold Nanoparticles.

C-reactive protein (CRP) levels can vary widely related to diverse disease contexts. However, expensive antibodies have impeded the clinical utility of antibody-based full-range CRP assays, especially in developing countries. Herein, we established a low-cost, antibody-free, 96-well plate-based full-range CRP detection method by combining gold nanoparticles (AuNPs), silver iodide (AgI), Eosin Y, and the aptamer hairpin probe (AHP) with Ag+-mediated cytosine-cytosine mismatches, that is, the Au@AgI/Eosin Y-AHP method. After binding the target CRP, the AHP released Ag+, which subsequently induced the aggregation of AuNPs on the surface of AgI colloids, resulting in a significant increase in the adsorption of Eosin Y on the surface of AuNPs. The changes in fluorescence intensity (FI) of Eosin Y in the supernate without and with CRP were proportional to the concentration of the CRP in the wide range of 0.01-40 ng/mL (r = 0.9969), and 96 samples can be detected in 96-well plates simultaneously by a microplate reader within 45 min. Remarkably, the CRP levels of 100 clinical samples achieved with the Au@AgI/Eosin Y-AHP had a good correlation with those obtained with the latex-enhanced immune turbidimetry assay (r = 0.986). Furthermore, the kit based on the Au@AgI/Eosin Y-AHP method costs only $8.1 for 100 tests. Therefore, the new method is beneficial for less developed areas where expensive assays are not affordable.