Yang Luo

Biomedical Applications of Terahertz Spectroscopy and Imaging

Terahertz (THz=10(12)Hz) radiation has attracted wide attention for its unprecedented sensing ability and its noninvasive and nonionizing properties. Tremendous strides in THz instrumentation have prompted impressive breakthroughs in THz biomedical research. Here, we review the current state of THz spectroscopy and imaging in various biomedical applications ranging from biomolecules, including DNA/RNA, amino acids/peptides, proteins, and carbohydrates, to cells and tissues. We also address the potential biological effects of THz radiation during its biological applications and propose future prospects for this cutting-edge technology.

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.

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 microg/L, with 100 microg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.

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.

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.

Rapid and simultaneous determination of essential minerals and trace elements in human milk by improved flame atomic absorption spectroscopy (FAAS) with microwave digestion.

A method for the simultaneous and economical determination of many trace elements in human milk is developed. Two multi-element hollow cathode lamps (HCLs) were used instead of single-element HCLs to improve the sample throughput of flame atomic absorption spectroscopy (FAAS). The microwave digestion of milk is optimized prior to detection, and the performance characteristics of the improved analysis method are identified. Clinical samples are detected by both FAAS and inductively coupled plasma-optical emission spectroscopy (ICP-OES) for methodology evaluation. Results reveal that the proposed FAAS with multi-element HCLs could determine six essential minerals and trace elements within 15 min. This method provides a linear analytical range of 0.01-10 mg L(-1). For Ca, Cu, Fe, Mg, Mn, and Zn, the limits of determination are 1.5, 3, 1.8, 2.2, 2.1, and 1.3 microg L(-1), respectively. The mean relative standard deviations (RSDs) of intra- and interassays are lower than 7%. Excellent operational characteristics of rapidity, simplicity, and economy make the proposed method a promising one for the quantification of trace elements in human milk in clinics of underdeveloped areas.

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.

A dye-assisted paper-based point-of-care assay for fast and reliable blood grouping

A paper-based assay for rapid and reliable blood grouping uses dye-based color changes as a visual readout to identify distinct blood components. Finding the right type Blood type matching is important for pregnancy, blood transfusion, and bone marrow transplantation. Zhang et al. developed a blood typing assay based on the color change that occurs when a common pH indicator dye reacts with blood. Red blood cells (RBCs) and plasma were separated from small volumes of whole, uncentrifuged blood samples using antibodies immobilized on paper test strips. The assays performed forward grouping (detecting A and/or B antigens on RBCs) and reverse grouping (monitoring the agglutination between RBCs and anti-A and/or anti-B antibodies in plasma) within 2 min and could also perform Rhesus and rare blood typing. A machine-learning algorithm grouped human blood samples automatically on the basis of spectral analysis of the colorimetric assay readouts. This economical and robust assay is useful for time- and resource-limited environments. Fast and simultaneous forward and reverse blood grouping has long remained elusive. Forward blood grouping detects antigens on red blood cells, whereas reverse grouping identifies specific antibodies present in plasma. We developed a paper-based assay using immobilized antibodies and bromocresol green dye for rapid and reliable blood grouping, where dye-assisted color changes corresponding to distinct blood components provide a visual readout. ABO antigens and five major Rhesus antigens could be detected within 30 s, and simultaneous forward and reverse ABO blood grouping using small volumes (100 μl) of whole blood was achieved within 2 min through on-chip plasma separation without centrifugation. A machine-learning method was developed to classify the spectral plots corresponding to dye-based color changes, which enabled reproducible automatic grouping. Using optimized operating parameters, the dye-assisted paper assay exhibited comparable accuracy and reproducibility to the classical gel-card assays in grouping 3550 human blood samples. When translated to the assembly line and low-cost manufacturing, the proposed approach may be developed into a cost-effective and robust universal blood-grouping platform.