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.

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.

Poly-l-Lysine-Modified Graphene Field-Effect Transistor Biosensors for Ultrasensitive Breast Cancer miRNAs and SARS-CoV-2 RNA Detection.

(Mi)RNAs are important biomarkers for cancers diagnosis and pandemic diseases, which require fast, ultrasensitive, and economical detection strategies to quantitatively detect exact (mi)RNAs expression levels. The novel coronavirus disease (SARS-CoV-2) has been breaking out globally, and RNA detection is the most effective way to identify the SARS-CoV-2 virus. Here, we developed an ultrasensitive poly-l-lysine (PLL)-functionalized graphene field-effect transistor (PGFET) biosensor for breast cancer miRNAs and viral RNA detection. PLL is functionalized on the channel surface of GFET to immobilize DNA probes by the electrostatic force. The results show that PGFET biosensors can achieve a (mi)RNA detection range of five orders with a detection limit of 1 fM and an entire detection time within 20 min using 2 µL of human serum and throat swab samples, which exhibits more than 113% enhancement in terms of sensitivity compared to that of GFET biosensors. The performance enhancement mechanisms of PGFET biosensors were comprehensively studied based on an electrical biosensor theoretical model and experimental results. In addition, the PGFET biosensor was applied for the breast cancer miRNA detection in actual serum samples and SARS-CoV-2 RNA detection in throat swab samples, providing a promising approach for rapid cancer diagnosis and virus screening.

Graphene oxide-graphene Van der Waals heterostructure transistor biosensor for SARS-CoV-2 protein detection.

The current outbreaking of the coronavirus SARS-CoV-2 pandemic threatens global health and has caused serious concern. Currently there is no specific drug against SARS-CoV-2, therefore, a fast and accurate diagnosis method is an urgent need for the diagnosis, timely treatment and infection control of COVID-19 pandemic. In this work, we developed a field effect transistor (FET) biosensor based on graphene oxide-graphene (GO/Gr) van der Waals heterostructure for selective and ultrasensitive SARS-CoV-2 proteins detection. The GO/Gr van der Waals heterostructure was in-situ formed in the microfluidic channel through π-π stacking. The developed biosensor is capable of SARS-CoV-2 proteins detection within 20 min in the large dynamic range from 10 fg/mL to 100 pg/mL with the limit of detection of as low as ∼8 fg/mL, which shows ∼3 × sensitivity enhancement compared with Gr-FET biosensor. The performance enhancement mechanism was studied based on the transistor-based biosensing theory and experimental results, which is mainly attributed to the enhanced SARS-CoV-2 capture antibody immobilization density due to the introduction of the GO layer on the graphene surface. The spiked SARS-CoV-2 protein samples in throat swab buffer solution were tested to confirm the practical application of the biosensor for SARS-CoV-2 proteins detection. The results indicated that the developed GO/Gr van der Waals heterostructure FET biosensor has strong selectivity and high sensitivity, providing a potential method for SARS-CoV-2 fast and accurate detection.

Attomolar-Level Ultrasensitive and Multiplex microRNA Detection Enabled by a Nanomaterial Locally Assembled Microfluidic Biochip for Cancer Diagnosis.

Non-invasive early diagnosis is of great significance in disease pathologic development and subsequent medical treatments, and microRNA (miRNA) detection has attracted critical attention in early cancer screening and diagnosis. High-throughput, sensitive, economic, and fast miRNA sensing platforms are necessary to realize the low-concentration miRNA detection in clinical diagnosis and biological studies. Here, we developed an attomolar-level ultrasensitive, rapid, and multiple-miRNA simultaneous detection platform enabled by nanomaterial locally assembled microfluidic biochips. This platform presents a large linear detection regime of 1 aM-10 nM, an ultralow detection limit of 0.146 aM with no amplification, a short detection time of 35 min with multiplex miRNA sensing capability, and a small sample volume consumption of 2 µL. The detection results of five miRNAs in real samples from breast cancer patients and healthy humans indicate its excellent capacity for practical applications in early cancer diagnosis. The proposed ultrasensitive, rapid, and multiple-miRNA detection microfluidic biochip platform is a universal miRNA detection approach and an important and valuable tool in early cancer screening and diagnosis as well as biological studies.