Microfluidic Biochips for Single-Cell Isolation and Single-Cell Analysis of Multiomics and Exosomes.

Single-cell multiomic and exosome analyses are potent tools in various fields, such as cancer research, immunology, neuroscience, microbiology, and drug development. They facilitate the in-depth exploration of biological systems, providing insights into disease mechanisms and aiding in treatment. Single-cell isolation, which is crucial for single-cell analysis, ensures reliable cell isolation and quality control for further downstream analyses. Microfluidic chips are small lightweight systems that facilitate efficient and high-throughput single-cell isolation and real-time single-cell analysis on- or off-chip. Therefore, most current single-cell isolation and analysis technologies are based on the single-cell microfluidic technology. This review offers comprehensive guidance to researchers across different fields on the selection of appropriate microfluidic chip technologies for single-cell isolation and analysis. This review describes the design principles, separation mechanisms, chip characteristics, and cellular effects of various microfluidic chips available for single-cell isolation. Moreover, this review highlights the implications of using this technology for subsequent analyses, including single-cell multiomic and exosome analyses. Finally, the current challenges and future prospects of microfluidic chip technology are outlined for multiplex single-cell isolation and multiomic and exosome analyses.

Efficient EVs separation and detection by an alumina-nanochannel-array-membrane integrated microfluidic chip and an antibody barcode biochip.

BACKGROUND: Small endosome-derived lipid nanovesicles (30-200 nm) are actively secreted by living cells and serve as pivotal biomarkers for early cancer diagnosis. However, the study of extracellular vesicles (EVs) requires isolation and purification from various body fluids. Although traditional EVs isolation and detection technologies are mature, they usually require large amount of sample, consumes long-time, and have relatively low-throughput. How to efficiently isolate, purify and detect these structurally specific EVs from body fluids with high-throughput remains a great challenge in in vitro diagnostics and clinical research. RESULTS: Herein, we suggest a nanosized microfluidic device for efficient and economical EVs filtration based on an alumina nanochannel array membrane. We evaluated the filtration device performance of alumina membranes with different diameters and found that an optimized chamber array with a hydrophilic-treated channel diameter of 90 nm could realize a filtration efficiency of up to 82% without any assistance from chemical or physical separation methods. Importantly, by integrating meticulously designed multichannel microfluidic biochips, EVs can be captured in-situ and monitored by antibody barcode biochip. The proposed filtration chip together with the high-throughput detection chip were capable of filtration of a few tens of µL samples and recognition of different phonotypes. The practical filtration and detection of EVs from clinical samples demonstrated the high performance of the device. SIGNIFICANT: Overall, this work provides a cost-effective, highly efficient and automated EVs filtration chip and detection dual-function integrated chip platform, which can directly separate EVs from serum or cerebrospinal fluid with an efficiency of 82% and conduct in-situ detection. This small fluidic device can provide a powerful tool for highly efficient identifying and analyzing EVs, presenting great application potential in clinical detection.

Self-assembled C-Ag hybrid nanoparticle on nanoporous GaN enabled ultra-high enhancement factor SERS sensor for sensitive thiram detection.

Considering pesticide residues cause significant harm to public health and the environment, developing a simple, sensitive, and reliable approach to pesticide residue detection to address this issue is necessary. In this study, an ultrasensitive and reliable surface-enhanced Raman scattering (SERS) sensor was developed using cetylpyridinium chloride as a protecting and reducing agent for the in situ synthesis and self-assembly of C-Ag nanoparticles on nanoporous GaN for the quantitative detection of thiram. A systematic investigation of the performance of the SERS sensor revealed that the SERS sensor delivered a limit of detection (LOD) of 10-14 M and an enhancement factor of up to 1.80 × 1011 with reasonable uniformity and reproducibility, with the stability of the SERS sensor demonstrated via long-term storage for up to 22 weeks in air. The enhancement mechanism of the SERS sensor was verified using a finite-difference time-domain simulation. The SERS sensor successfully detected thiram in real samples with an LOD of 10-10 M. Hence, this study provides an effective platform for monitoring food safety and the environment.

A novel diagnostic signature of circulating tsRNAs and miRNAs in esophageal squamous cell carcinoma detected with a microfluidic platform.

Small non-coding RNAs (sncRNAs) consisting of tRNA-derived small RNAs (tsRNAs) and miRNAs can be released by cancer cells and detected in blood, offering great potential for diagnosis of malignant tumors such as squamous cell carcinoma of the esophagus (ESCC). One of the major challenges for the clinical application of blood-based sncRNAs biomarkers is the difficulty of detection because of their small sncRNA size and low abundance. The deferentially expressed tsRNAs and miRNAs in plasma were studied with high-throughput sequencing and polymerase chain reaction in ESCC cohorts. A novel signature containing tRF-55:74-chrM.Phe-GAA, tRF-56:75-Ala-CGC-1-M4 and miR-4488 was identified with diagnostic potential. The signature was further confirmed by an attomolar-level ultrasensitive and rapid microfluidic biochip, which can achieve a multiplex, simple and low-cost detection. Our results indicated that a combination of tsRNAs and miRNAs has high diagnostic efficiency and tremendous potential to act as specific biomarkers through a reliable, highly sensitive, fast, and economic microfluidic biochip for ESCC diagnosis.

A self-assembly hydrophobic oCDs/Ag nanoparticles SERS sensor for ultrasensitive melamine detection in milk.

A simple, ultra-sensitive, and super-stable hydrophobic SERS platform for detection of melamine in milk is developed. The hydrophobic SERS platform was constructed via directly growing hydrophobic carbon/silver nanoparticles on glass by in-situ one-step carbonization using hexadecylpyridinium chloride monohydrate as stabilizer and reducing agent. The performances of SERS platform are systematically studied by using Rhodamine 6G (R6G) as a model, which achieves detection level of 10-13 M and enhancement factor of 3.4 × 1010 for R6G detection with good uniformity and reproducibility, as well as 110 days stability in air. The FDTD simulation was used to confirm SERS enhancement mechanism. More importantly, SERS platform delivers good linear property in the range from 0.01 to 100 ppm, and low limit detection of 9 ppb for melamine detection in milk through direct drop on the platform. The SERS platform could have great applications in food safety, environmental monitoring, biomedicine and other fields.

A Sensitive and Portable Double-Layer Microfluidic Biochip for Harmful Algae Detection.

Harmful algal blooms (HABs) are common disastrous ecological anomalies in coastal waters. An effective algae monitoring approach is important for natural disaster warning and environmental governance. However, conducting rapid and sensitive detection of multiple algae is still challenging. Here, we designed an ultrasensitive, rapid and portable double-layer microfluidic biochip for the simultaneous quantitative detection of six species of algae. Specific DNA probes based on the 18S ribosomal DNA (18S rDNA) gene fragments of HABs were designed and labeled with the fluorescent molecule cyanine-3 (Cy3). The biochip had multiple graphene oxide (GO) nanosheets-based reaction units, in which GO nanosheets were applied to transfer target DNA to the fluorescence signal through a photoluminescence detection system. The entire detection process of multiple algae was completed within 45 min with the linear range of fluorescence recovery of 0.1 fM-100 nM, and the detection limit reached 108 aM. The proposed approach has a simple detection process and high detection performance and is feasible to conduct accurate detection with matched portable detection equipment. It will have promising applications in marine natural disaster monitoring and environmental care.

High-Throughput, Living Single-Cell, Multiple Secreted Biomarker Profiling Using Microfluidic Chip and Machine Learning for Tumor Cell Classification.

Secreted proteins provide abundant functional information on living cells and can be used as important tumor diagnostic markers, of which profiling at the single-cell level is helpful for accurate tumor cell classification. Currently, achieving living single-cell multi-index, high-sensitivity, and quantitative secretion biomarker profiling remains a great challenge. Here, a high-throughput living single-cell multi-index secreted biomarker profiling platform is proposed, combined with machine learning, to achieve accurate tumor cell classification. A single-cell culture microfluidic chip with self-assembled graphene oxide quantum dots (GOQDs) enables high-activity single-cell culture, ensuring normal secretion of biomarkers and high-throughput single-cell separation, providing sufficient statistical data for machine learning. At the same time, the antibody barcode chip with self-assembled GOQDs performs multi-index, highly sensitive, and quantitative detection of secreted biomarkers, in which each cell culture chamber covers a whole barcode array. Importantly, by combining the K-means strategy with machine learning, thousands of single tumor cell secretion data are analyzed, enabling tumor cell classification with a recognition accuracy of 95.0%. In addition, further profiling of the grouping results reveals the unique secretion characteristics of subgroups. This work provides an intelligent platform for high-throughput living single-cell multiple secretion biomarker profiling, which has broad implications for cancer investigation and biomedical research.

Lys-AuNPs@MoS2 Nanocomposite Self-Assembled Microfluidic Immunoassay Biochip for Ultrasensitive Detection of Multiplex Biomarkers for Cardiovascular Diseases.

The progression of cardiovascular diseases is accompanied by myocardial injury and necrosis, heart failure, and inflammatory response. Accordingly, ultrasensitive and rapid detection of multiple biomarkers plays a vital role in clinical diagnosis and timely treatment. Here, we developed a novel Lys-AuNPs@MoS2 nanocomposite self-assembled microfluidic immunoassay biochip with digital signal output and applied it to the simultaneous detection of multiple serum biomarkers including inflammatory factors and cardiovascular biomarkers, PCT, CRP, IL6, cTnI, cTnT, and NT-BNP, with high throughput and sensitivity. The digital output signal was collected in the solid phase on the chip surface with two-dimensional distribution of targets. Lys-AuNPs@MoS2 nanocomposites self-assembled biochips could simultaneously detect all six biomarkers in 60 samples in 40 min with detection limit of a few to tens of pg/mL for all serum biomarkers. The microfluidic biochip based on Lys-AuNPs@MoS2 nanocomposites provides a promising method in applications for clinical diagnosis.

Rapid and High-Throughput SARS-CoV-2 RNA Detection without RNA Extraction and Amplification by Using a Microfluidic Biochip.

The ongoing outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has spread globally and poses a threat to public health and National economic development. Rapid and high-throughput SARS-CoV-2 RNA detection without the need of RNA extraction and amplification remain a key challenge. In this study, a new SARS-CoV-2 RNA detection strategy using a microfluidic biochip for the rapid and ultrasensitive detection of SARS-CoV-2 without RNA extraction and amplification was developed. This new strategy takes advantage of the specific SARS-CoV-2 RNA and probe DNA reaction in the microfluidic channel, fluorescence signal regulation by nanomaterials, and accurate sample control by the microfluidic chip. It presents an ultralow limit of detection of 600 copies mL-1 in a large linear detection regime from 1 aM to 100 fM. Fifteen samples were simultaneously detected in 40 min without the need for RNA purification and amplification. The detection accuracy of the strategy was validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR), with a recovery of 99-113 %. Therefore, the SARS-CoV-2 RNA detection strategy proposed in this study can potentially be used for the quantitative diagnosis of viral infectious diseases.

Ultrasensitive, high-throughput, and rapid simultaneous detection of SARS-CoV-2 antigens and IgG/IgM antibodies within 10 min through an immunoassay biochip.

COVID-19 is now a severe threat to global health. Facing this pandemic, we developed a space-encoding microfluidic biochip for high-throughput, rapid, sensitive, simultaneous quantitative detection of SARS-CoV-2 antigen proteins and IgG/IgM antibodies in serum. The proposed immunoassay biochip integrates the advantages of graphene oxide quantum dots (GOQDs) and microfluidic chip and is capable of conducting multiple SARS-CoV-2 antigens or IgG/IgM antibodies of 60 serum samples simultaneously with only 2 µL sample volume of each patient. Fluorescence intensity of antigens and IgG antibody detection at emission wavelength of ~680 nm was used to quantify the target concentration at excitation wavelength of 632 nm, and emission wavelength of ~519 nm was used during the detection of IgM antibodies at excitation wavelength of 488 nm. The method developed has a large linear quantification detection regime of 5 orders of magnitude, an ultralow detection limit of ~0.3 pg/mL under optimized conditions, and less than 10-min qualitative detection time. The proposed biosensing platform will not only greatly facilitate the rapid diagnosis of COVID-19 patients, but also provide a valuable screening approach for infected patients, medical therapy, and vaccine recipients.

Colorimetric detection of Ba2+, Cd2+ and Pb2+ based on a multifunctionalized Au NP sensor.

In this work, we developed a colorimetric method for the detection of three kinds of ions with one kind of detection reagent. In detail, gold nanoparticles (Au NPs) multifunctionalized with 3-mercaptonicotinic acid and 4-aminobenzo-18-crown-6 (3-MPA-abc) were prepared and used as a colorimetric sensor for the simple and rapid detection of Ba2+, Cd2+ and Pb2+ ions. After adding Ba2+/Cd2+/Pb2+, the oxygen atom in the crown ether cavity and the carboxyl group of 3-mercaptopropionic acid can react with Ba2+/Cd2+/Pb2+ to form coordination bonds, resulting in the aggregation of the functionalized Au NPs and the color change of Au NP solution. The LOD of the colorimetric sensor for Ba2+/Cd2+/Pb2+ is 20 nM, 20 nM and 50 nM by the naked eye, respectively. A good linear relationship (R2 = 0.9984, R2 = 0.9917, R2 = 0.9934) between the absorbance ratio and Ba2+/Cd2+/Pb2+ concentrations indicates that our Au NP based colorimetric sensor can be used for the quantitative assay of Ba2+/Cd2+/Pb2+, and this detection method was successfully applied in the detection of Ba2+/Cd2+/Pb2+ in real environmental samples.