Tianlun Jiang

Sensitive and rapid quantification of C-reactive protein using quantum dot-labeled microplate immunoassay

BackgroundHigh-sensitivity C-reactive protein (hs-CRP) assay is of great clinical importance in predicting risks associated with coronary heart disease. Existing hs-CRP assays either require complex operation or have low throughput and cannot be routinely implemented in rural settings due to limited laboratory resources.MethodsWe developed a novel hs-CRP assay capable of simultaneously quantifying over 90 clinical samples by using quantum dots-labeled immunoassay within a standard 96-well microplate. The specificity of the assay was enhanced by adopting two monoclonal antibodies (mAbs) that target distinct hs-CRP epitopes, serving as the coating antibody and the detection antibody, respectively. In the presence of hs-CRP antigen, the fluorescence intensity of the mAb-Ag-mAb sandwich complex captured on the microplate can be read out using a microplate reader.ResultsThe proposed hs-CRP assay provides a wide analytical range of 0.001-100 mg/L with a detection limit of 0.06 (0.19) μg/L within 1.5 h. The accuracy of the proposed assay has been confirmed for low coefficient of variations (CVs), 2.27% (intra-assay) and 8.52% (inter-assay), together with recoveries of 96.7-104.2%. Bland-Altman plots of 104 clinical samples exhibited good consistency among the proposed assay, commercial high-sensitivity ELISA, and nephelometry, indicating the prospects of the newly developed hs-CRP assay as an alternative to existing hs-CRP assays.ConclusionThe developed assay meets the needs of the rapid, sensitive and high-throughput determination of hs-CRP levels within a short time using minimal resources. In addition, the developed assay can also be used to detect and quantify other diagnostic biomarkers by immobilizing specific monoclonal antibodies.

Dynamic Monitoring of MicroRNA–DNA Hybridization Using DNAase-Triggered Signal Amplification

Dynamically monitoring microRNA (miRNA)-DNA reactions is critical for elucidating various biological processes. However, traditional strategies fail to capture this dynamic event because the original targets are preamplified. In the present study, we developed an amplification-free strategy for real-time monitoring of miRNA-DNA hybridization that integrates the advantages of both duplex-specific nuclease (DSN)-triggered signal amplification and single-stranded DNA probe coating facilitated by reduced graphene oxide. DSN-mediated miRNA recognition was found to consist of two phases: hybridization and hybridization cleavage. In the presence of miRNA and DSN, hybridization of a 22-mer miRNA-DNA could be completed within 7 min by observing the angle increase in a surface plasmon resonance (SPR) biosensor. The subsequent hybridization-cleavage process could be visualized as a gradual SPR angle decrease that occurred until all coated probes were hydrolyzed. In addition, for miRNA-21 detection, the proposed linear signal amplification assay demonstrated a sensitivity of 3 fM over a dynamic range of 5 orders of magnitude.

Duplex-specific nuclease-mediated bioanalysis

Duplex-specific nucleases (DSNs) are promising tools for bioanalysis because of their unique ability to cleave DNA within duplexes while keeping a single strand intact. There is prevalent use of DSNs in both biomedical and biological science applications, such as cDNA library construction, circulating miRNA detection, telomeric overhang detection, and SNP recognition. We present an overview of the current knowledge of DSNs, with special emphasis on DSN-mediated isothermal signal amplification strategies for trace miRNA detection. Continued innovation to address key challenges, such as amplification-free approaches, will open up new avenues in the field of miRNA profiling, offering opportunities for improved personalized medicine, preventive medicine, and translational medicine.

Visible light mediated killing of multidrug-resistant bacteria using photoacids

Increasing acidity is a promising method for bacterial inactivation by inhibiting the synthesis of intracellular proteins at low pH. However, conventional ways of pH control are not reversible, which can cause continuous changes in cellular and biological behaviours and are harmful to the host. Utilizing a photoacid that can reversibly alter pH over two units, we demonstrated a strong bacterial inhibition assisted by visible light. The pH value of the solution reverts back to the original level immediately after the irradiation is stopped. If a photoacid is combined with colistin, the minimum inhibitory concentration (MIC) of colistin on multidrug-resistant (MDR) Pseudomonas aeruginosa can be improved ∼32 times (from 8 to 0.25 μg mL-1), which significantly decreases the toxicity of colistin in clinics. Evidenced by the extremely low toxicity of the photoacid, this strategy is promising in MDR bacteria killing.

Dual-aptamer-based biosensing of toxoplasma antibody.

A panel of seven aptamers to antitoxoplasma IgG is first discovered in this report. The aptamers are selected using systematic evolution of ligands by exponential enrichment (SELEX) technology, cloned, and identified by sequencing and affinity assay. Among them, two aptamers (TGA6 and TGA7) with the highest affinities are employed as capture probe and detection probe in developing a quantum dots-labeled dual aptasensor (Q-DAS). In the presence of antitoxoplasma IgG, an aptamer-protein-aptamer sandwich complex (TGA6-IgG-TGA7) is formed and captured on a multiwell microplate, whose fluorescence can be read out using quantum dots as the fluorescence label, ensuring highly sensitive and specific sensing of antitoxoplasma IgG. The operating characteristics of the proposed assay are guaranteed using dual aptamers as the recognizing probes when compared with antibody-based immunoassay. Q-DAS has a linearity within the range of 0.5-500 IU with a lowest detection of 0.1 IU. Receiver operating curves of 212 clinical samples show a 94.8% sensitivity and 95.7% specificity when the cutoff value is set as 6.5 IU, indicating the proposed Q-DAS is a promising assay in large-scale screening of toxoplasmosis.

Rapid label-free identification of mixed bacterial infections by surface plasmon resonance

BackgroundEarly detection of mixed aerobic-anaerobic infection has been a challenge in clinical practice due to the phenotypic changes in complex environments. Surface plasmon resonance (SPR) biosensor is widely used to detect DNA-DNA interaction and offers a sensitive and label-free approach in DNA research.MethodsIn this study, we developed a single-stranded DNA (ssDNA) amplification technique and modified the traditional SPR detection system for rapid and simultaneous detection of mixed infections of four pathogenic microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium tetani and Clostridium perfringens).ResultsWe constructed the circulation detection well to increase the sensitivity and the tandem probe arrays to reduce the non-specific hybridization. The use of 16S rDNA universal primers ensured the amplification of four target nucleic acid sequences simultaneously, and further electrophoresis and sequencing confirmed the high efficiency of this amplification method. No significant signals were detected during the single-base mismatch or non-specific probe hybridization (P < 0.05). The calibration curves of amplification products of four bacteria had good linearity from 0.1 nM to 100 nM, with all R2 values of >0.99. The lowest detection limits were 0.03 nM for P. aeruginosa, 0.02 nM for S. aureus, 0.01 nM for C. tetani and 0.02 nM for C. perfringens. The SPR biosensor had the same detection rate as the traditional culture method (P < 0.05). In addition, the quantification of PCR products can be completed within 15 min, and excellent regeneration greatly reduces the cost for detection.ConclusionsOur method can rapidly and accurately identify the mixed aerobic-anaerobic infection, providing a reliable alternative to bacterial culture for rapid bacteria detection.

Interference-free determination of ischemia-modified albumin using quantum dot coupled X-ray fluorescence spectroscopy.

Ischemia-modified protein (IMA) is the most sensitive diagnostic biomarker of ischemic heart disease, but differentiation of IMA from human serum albumin (HSA), a ubiquitous serum protein, is still challenging owing to the shared antigenicity. In this investigation, we developed a rapid and interference-free approach for IMA determination using quantum dots-coupled X-ray Fluorescence Spectroscopy (Q-XRF). In a typical Q-XRF assay, serum total HSA is quantified using quantum dot-coupled sandwich immunoassay, and intact HSA (iHSA) is determined using a XRF spectroscopy, by measuring XRF intensity of Co (II) bonded to iHSA. IMA concentration is automatically determined within 30 min by calculating the difference between total HSA and iHSA. This strategy can effectively eliminate the interference from native HSA level. Results show that no significant influences have been observed from hemolysis or high levels of cholesterol (7 mg/L), triglyceride (5.2 mg/L), IgG (10 g/L), and fibrinogen (4 g/L). A linearity of 1-100mg/mL is obtained in iHSA determination using XRF (r(2)=0.979). The proposed Q-XRF assay demonstrates a lowest detection limit of 0.05 U/mL. Receiver-operating characteristic (ROC) curves reveal that Q-XRF assay provide an improved sensitivity than ACB assay (95.9% vs. 82.9%) in differentiating ischemic patients from health individuals, at an optimal cutoff point of 79.2U/mL. The proposed approach provides a new strategy for interference-free, simple and rapid evaluation of IMA concentration by combining sandwich immunoassay and XRF spectroscopy.