Predictive Analysis in Oral Cancer Immunotherapy: Profiling Dual PD-L1-Positive Extracellular Vesicle Subtypes with Step-Wedge Microfluidic Chips.

PD-L1-positive extracellular vesicles (PD-L1+ EVs) play a pivotal role as predictive biomarkers in cancer immunotherapy. These vesicles, originating from immune cells (I-PD-L1+ EVs) and tumor cells (T-PD-L1+ EVs), hold distinct clinical predictive values, emphasizing the importance of deeply differentiating the PD-L1+ EV subtypes for effective liquid biopsy analyses. However, current methods such as ELISA lack the ability to differentiate their cellular sources. In this study, a novel step-wedge microfluidic chip that combines magnetic microsphere separation with single-layer fluorescence counting is developed. This chip integrates magnetic microspheres modified with anti-PD-L1 antibodies and fluorescent nanoparticles targeting EpCAM (tumor cell marker) or CD45 (immunocyte marker), enabling simultaneous quantification and sensitive analysis of PD-L1+ EV subpopulations in oral squamous cell carcinoma (OSCC) patients' saliva without background interference. Analysis results indicate reduced levels of I-PD-L1+ EVs in OSCC patients compared to those in healthy individuals, with varying levels of heterogeneous PD-L1+ EVs observed among different patient groups. During immunotherapy, responders exhibit decreased levels of total PD-L1+ EVs and T-PD-L1+ EVs, accompanied by reduced levels of I-PD-L1+ EVs. Conversely, nonresponders show increased levels of I-PD-L1+ EVs. Utilizing the step-wedge microfluidic chip allows for simultaneous detection of PD-L1+ EV subtypes, facilitating the precise prediction of oral cancer immunotherapy outcomes.

Magnetic Microbead-Based Herringbone Chip for Sensitive Detection of Human Immunodeficiency Virus.

The microfluidic chip-based nucleic acid detection method significantly improves the sensitivity since it precisely controls the microfluidic flow in microchannels. Nonetheless, significant challenges still exist in improving the detection efficiency to meet the demand for rapid detection of trace substances. This work provides a novel magnetic herringbone (M-HB) structure in a microfluidic chip, and its advantage in rapid and sensitive detection is verified by taking complementary DNA (cDNA) sequences of human immunodeficiency virus (HIV) detection as an example. The M-HB structure is designed based on controlling the magnetic field distribution in the micrometer scale and is formed by accumulation of magnetic microbeads (MMBs). Hence, M-HB is similar to a nanopore microstructure, which has a higher contact area and probe density. All of the above is conducive to improving sensitivity in microfluidic chips. The M-HB chip is stable and easy to form, which can linearly detect cDNA sequences of HIV quantitatively ranging from 1 to 20 nM with a detection limit of 0.073 nM. Compared to the traditional herringbone structure, this structure is easier to form and release by controlling the magnetic field, which is flexible and helps in further study. Results show that this chip can sensitively detect the cDNA sequences of HIV in blood samples, demonstrating that it is a powerful platform to rapidly and sensitively detect multiple nucleic acid-related viruses of infectious diseases.

Simultaneous detection of multiple influenza virus subtypes based on microbead-encoded microfluidic chip.

Influenza virus, existing many subtypes, causes a huge risk of people health and life. Different subtypes bring a huge challenge for detection and treatment, thus simultaneous detection of multiple influenza virus subtypes plays a key role in fight against this disease. In this work, three kinds of influenza virus subtypes are one-step detection based on microbead-encoded microfluidic chip. HIN1, H3N2 and H7N3 were simultaneously captured only by microbeads of different magnetism and sizes, and they were further treated by magnetic separation and enriched through the magnetism and size-dependent microfluidic structure. Different subtypes of influenza virus could be linearly encoded in different detection zones of microfluidic chip according to microbeads of magnetism and size differences. With the high-brightness quantum dots (QDs) as label, the enriched fluorescence detection signals were further read online from linearly encoded strips, obtaining high sensitivity with detection limit of HIN1, H3N2, H7N3 about 2.2 ng/mL, 3.4 ng/mL and 2.9 ng/mL. Moreover, a visual operation interface, microcontroller unit and two-way syringe pump were consisted of a miniaturized detection device, improving the detection process automation. And this assay showed strong specificity. This method improves a new way of multiple pathogens detection using microbead-encoded technologies in the microfluidic chip.

Continuous magnetic separation microfluidic chip for tumor cell in vivo detection.

Continuously recording the dynamic changes of circulating tumor cells (CTCs) is crucial for tumor metastasis. This paper creates a continuous magnetic separation microfluidic chip that enables rapid and continuous in vivo cell detection. The chip shows its potential to study tumor cell circulation in the blood, offering a new platform for studying the cellular mechanism of tumor metastasis.

Magnetic-based Microfluidic Chip: A Powerful Tool for Pathogen Detection and Affinity Reagents Selection.

The global outbreak of pathogen diseases has brought a huge risk to human lives and social development. Rapid diagnosis is the key strategy to fight against pathogen diseases. Development of detection methods and discovery of related affinity reagents are important parts of pathogen diagnosis. Conventional detection methods and affinity reagents discovery have some problems including much reagent consumption and labor intensity. Magnetic-based microfluidic chip integrates the unique advantages of magnetism and microfluidic technology, improving a powerful tool for pathogen detection and their affinity reagent discovery. This review provides a summary about the summary of pathogen detection through magnetic-based microfluidic chip, which refers to the pathogen nucleic acid detection (including extraction, amplification and signal acquisition), pathogen proteins and antibodies detection. Meanwhile, affinity reagents are served as the critical tool to specially capture pathogens. New affinity reagents are discovered to further facilitate the pathogen diagnosis. Microfluidic technology has also emerged as a powerful tool for affinity reagents discovery. Thus, this review further introduced the selection progress of aptamer as next generation affinity through the magnetic-based microfluidic technology. Using this selection technology shows great potential to improve selection performance, including integration and highly efficient selection. Finally, an outlook is given on how this field will develop on the basis of ongoing pathogen challenges.

Precise and convenient size barcode on microfluidic chip for multiplex biomarker detection.

The existing multiplex biomarker detection methods are limited by the high demand for coding material and expensive detection equipment. This paper proposes a convenient and precise coding method based on a wedge-shaped microfluidic chip, which can be further applied in multiplex biomarker detection. The proposed microfluidic chip has a microchannel with continuously varying height, which can naturally separate and code microparticles of different sizes. Our data indicate that this method can be applied to code more than 5 or 7 kinds of microparticles, even when their size discrepancies are smaller than 1 µm. Based on these, multiplex biomarker detection can be implemented by using microparticles of different sizes, hence each kind of microparticle that coats one kind of antibody represents the species of targets. This method is simple and easy to operate, with no clogging or sophisticated coding design, showing its significant potential in the area of point-of-care tests (POCT).

An automated detection of influenza virus based on 3-D magnetophoretic separation and magnetic label.

Automated detection of the influenza virus is important for the prevention of infectious viruses. Herein, assisted by three-dimensional (3-D) magnetophoretic separation and magnetic label, an automated detection device was constructed for H7N9 influenza virus hemagglutinin. Multi-layer glass slides were used to generate a 3-D microchannel network with two-level channels, realizing 3-D magnetophoretic separation with a magnetic field in the vertical direction to microchannels for the sample treatment. After the immunomagnetic separation, a magnetic-tagged complex was captured in an antibody-modified glass capillary, where magnetic beads further as a label could cause the voltage change of the miniature tube liquid sensor to obtain the detection signal. Moreover, the whole detection process and detection results were controlled and read through a liquid crystal display (LCD) screen to improve the automation. Finally, the detection limit was calculated to be 8.4 ng mL-1 for H7N9 hemagglutinin and had good specificity and reproducibility. These results indicate that this detection device proposes promising automated avenues for the early detection of infectious diseases.

Negative depletion mediated brightfield circulating tumour cell identification strategy on microparticle-based microfluidic chip.

BACKGROUND: The most convenient circulating tumor cells (CTCs) identification method is direct analysis of cells under bright field microscopy by which CTCs can be comprehensive studied based on morphology, phenotype or even cellular function. However, universal cell markers and a standard tumour cell map do not exist, thus limiting the clinical application of CTCs. RESULTS: This paper focuses on an automatic and convenient negative depletion strategy for circulating tumour cell identification under bright field microscopy. In this strategy, immune microparticles (IMPs) are applied to negatively label white blood cells rather than the tumour cells, such that tumour cells can be directly distinguished under brightfield of the microscopy. In this way, all of the heterogeneous tumour cells and their phenotype properties can be retained for further cancer-related studies. In addition, a wedge-shaped microfluidic chip is constructed for heterogeneous CTC pre-purification and enrichment by size, thus significantly decreasing the interference of haematological cells. Additionally, all cell treatments are processed automatically, and the tumour cells can be rapidly counted and distinguished via customized cell analytical software, showing high detection efficiency and automation. This IMPs based negative cell labelling strategy can also be combined with other classic cell identification methods, thus demonstrating its excellent compatibility. CONCLUSION: This identification strategy features simple and harmless for tumour cells, as well as excellent accuracy and efficiency. And the low equipment demand and high automation level make it promise for extensive application in basic medical institutions.

Simultaneous and automated detection of influenza A virus hemagglutinin H7 and H9 based on magnetism and size mediated microfluidic chip.

Influenza viruses with multiple subtypes have highly virulent in humans, of which influenza hemagglutinin (HA) is the major viral surface antigen. Simultaneous and automated detection of multiple influenza HA are of great importance for early-stage diagnosis and operator protection. Herein, a magnetism and size mediated microfluidic platform was developed for point-of-care detection of multiple influenza HA. With multiplex microvalves and computer program control, the detection process showed high automation which had a great potential for avoiding the high-risk virus exposure to the operator. Taking advantage of magnetism and size mediated multiple physical fields, multiple influenza HA could be simultaneous separation and detection depended on different-size magnetic beads. Using high-luminance quantum dots as reporter, this assay achieved high sensitivity with a detection limit of 3.4 ng/mL for H7N9 HA and 4.5 ng/mL for H9N2 HA, and showed excellent specificity, anti-interference ability and good reproducibility. These results indicate that this method may propose new avenues for early detection of multiple influenza subtypes.

High-performance multiplex microvalves fabrication and using for tumor cells staining on a microfluidic chip.

As we all know, microvalve holds great importance for microfluidic manipulation in chip. Herein, a simple method of high-performance multiplex microvalves chip fabrication was reported. In this method, a sandwich structure is established by inserting a polydimethylsiloxane (PDMS) membrane into two glasses, which is cheap and simple without any complex silicon-based device or soft lithography. Taking advantages of both the elasticity of the PDMS and the rigidity of glass, the microvalve chip showed good controls performance and had the ability of multiplex integration. Moreover, aided by a computer design program, this microvalves chip can be performed automatically, showing great potential to develop new highly integrated microfluidic devices. In addition, the fabricated multiplex microvalve chip is further successfully used for staining tumor cells automatically, improving the efficiency of cell identification process and reducing human errors. These results indicate this method opens up new avenues for multiplex microvalves fabrication and its biological application.

Ebola Virus Aptamers: From Highly Efficient Selection to Application on Magnetism-Controlled Chips.

Aptamers for Ebola virus (EBOV) offer a powerful means for prevention and diagnostics. Unfortunately, few aptamers for EBOV have been discovered yet. Herein, assisted by magnetism-controlled selection chips to strictly manipulate selection conditions, a highly efficient aptamer selection platform for EBOV is proposed. With highly stringent selection conditions of rigorous washing, manipulation of minuscule amounts of magnetic beads, and real-time evaluation of the selection effectiveness, the selection performance of the platform was improved significantly. In only three rounds of selection, the high-performance aptamers for EBOV GP and NP proteins were obtained simultaneously, with dissociation constants ( Kd) in the nanomolar range. The aptamer was further applied to the detection of EBOV successfully, with a detection limit of 4.2 ng/mL. The whole detection process that consisted of sample mixing, separation, and signal acquisition was highly integrated and conducted in a magnetism-controlled detection chip, showing high biosafety and great potential for point-of-care detection. The method may open up new avenues for prevention and control of EBOV.

A simple pyramid-shaped microchamber towards highly efficient isolation of circulating tumor cells from breast cancer patients.

Isolation and detection of circulating tumor cells (CTCs) has showed a great clinical impact for tumor diagnosis and treatment monitoring. Despite significant progresses of the existing technologies, feasible and cost-effective CTC isolation techniques are more desirable. In this study, a novel method was developed for highly efficient isolation of CTCs from breast cancer patients based on biophysical properties using a pyramid-shaped microchamber. Through optimization tests, the outlet height of 6 µm and the flow rate of 200 µL/min were chosen as the optimal conditions. The capture efficiencies of more than 85% were achieved for cancer cell lines (SKBR3, BGC823, PC3, and H1975) spiked in DMEM and healthy blood samples without clogging issue. In clinic assay, the platform identified CTCs in 13 of 20 breast cancer patients (65%) with an average of 4.25 ± 4.96 CTCs/2 mL, whereas only one cell was recognized as CTC in 1 of 15 healthy blood samples. The statistical analyses results demonstrated that both CTC positive rate and CTC counts were positive correlated with TNM stage (p < 0.001; p = 0.02, respectively). This microfluidic platform successfully demonstrated the clinical feasibility of CTC isolation and would hold great potential of clinical application in predicting and monitoring the prognosis of cancer patients.

Cellular-Beacon-Mediated Counting for the Ultrasensitive Detection of Ebola Virus on an Integrated Micromagnetic Platform.

Ebola virus (EBOV) disease is a complex zoonosis that is highly virulent in humans and has caused many deaths. Highly sensitive detection of EBOV is of great importance for early-stage diagnosis for increasing the probability of survival. Herein, we established a cellular-beacon-mediated counting strategy for an ultrasensitive EBOV assay on a micromagnetic platform. The detection platform, which was assisted by on-demand magnetic-field manipulation, showed high integration and enhanced complex-sample pretreatment by magnetophoretic separation and continuous-flow washing. Cellular beacons (i.e., fluorescent cells) with superior optical properties were used as reporters, and each cellular beacon was used as a fluorescent tracking unit to quantify EBOV by counting the numbers of individual fluorescent signals on the micromagnetic platform. This method achieves high sensitivity with a detection limit as low as 2.6 pg/mL, and the detection limit shows little difference in a complex matrix. In addition, it has excellent specificity and good reproducibility. These results indicate that this method proposes an ultrasensitive detection strategy for early diagnosis of the disease.

Rapid detection and subtyping of multiple influenza viruses on a microfluidic chip integrated with controllable micro-magnetic field.

Influenza viruses have threatened animals and public health systems continuously. Moreover, there are many subtypes of influenza viruses, which have brought great difficulties to the classification of influenza viruses during any influenza outbreak. So it is crucial to develop a rapid and accurate method for detecting and subtyping influenza viruses. In this work, we reported a rapid method for simultaneously detecting and subtyping multiple influenza viruses (H1N1, H3N2 and H9N2) based on nucleic acid hybridization on a microfluidic chip integrated with controllable micro-magnetic field. H1N1, H3N2 and H9N2 could be simultaneously detected in 80min with detection limits about 0.21nM, 0.16nM, 0.12nM in order. Moreover, the sample and reagent consumption was as low as only 3µL. The results indicated that this approach possessed fast analysis and high specificity. Therefore, it is expected to be used to simultaneously subtype and detect multiple targets, and may provide a powerful technique platform for the rapid detection and subtyping analysis of influenza viruses.

A colorimetric and electrochemical immunosensor for point-of-care detection of enterovirus 71.

Point-of-care detection of human enterovirus 71 (EV71), the major pathogen that causes hand, foot, and mouth disease (HFMD) among children, is urgently needed for early diagnosis and control of related epidemics. A colorimetric and electrochemical immunosensor for point-of-care detection of EV71 has been developed based on dual-labeled magnetic nanobeads amplification. The dual-labeled magnetic nanobeads (DL-MBs) are fabricated by simultaneous immobilization of EV71 monoclonal antibody (mAb) and horseradish peroxidase (HRP) on magnetic nanobeads. By capturing EV71 virions in 20µL sample on mAb modified AuNPs-coated ITO electrode and subsequently binding with DL-MBs, with the addition of TMB and H2O2, colorimetric signals corresponding to EV71 with a concentration of 1.0ngmL-1 can be directly read out by naked eyes, making it possible towards point-of-care detection of the virus. Furthermore, on the reduction of oxidized TMB on the electrode, electrochemical signal can be detected in the same detection cell without solution transfer, with a detection limit of 0.01ngmL-1. Validated with clinical samples, the colorimetric and electrochemical immunosensor shows a complete consistency with reverse transcription-polymerase chain reaction (RT-PCR) results. So far as we know, this is the first report on EV71 detection using electrochemical method. The merits of this assay, including high sensitivity, ability for colorimetric detection and easy to operation, ensure a promising future in point-of-care diagnostics of virus related diseases.

Multifunctional Screening Platform for the Highly Efficient Discovery of Aptamers with High Affinity and Specificity.

Aptamers have attracted much attention as the next generation of affinity reagents. Unfortunately, the selection efficiency remains a critical bottleneck for the widespread application of aptamers. Herein, to accelerate aptamers discovery, a multifunctional microfluidic selection platform was developed, on which the selection efficiency was greatly improved and high-affinity and -specificity aptamers were generated within two round selections. The multifunctional screening platform, precisely manipulating magnetic beads on the micrometer scale, improved selection performance based on microfluidic continuous flow and enhanced the selection process control via in situ monitoring and real-time evaluation. This method could suppress ∼50-fold nonspecific binding nucleic acids compared to the conventional methods, further eliminate weakly bound nucleic acids within 9 min, and simultaneously perform the negative selection and positive selection. And the selection effectiveness was in situ and real-time monitoring. Three aptamers showed high affinity and specificity toward mucin 1 (MUC1) with dissociation constants (Kd) in nanomolar range (from 22 to 65 nM). Furthermore, the selected aptamer was able to specially label cancer cells and efficiently capture exosomes with 64% capture efficiency. It demonstrated that the multifunctional screening platform was an efficient method to generate high-quality aptamers in a rapid and economic manner.

A chip assisted immunomagnetic separation system for the efficient capture and in situ identification of circulating tumor cells.

The detection of circulating tumor cells (CTCs), a kind of "liquid biopsy", represents a potential alternative to noninvasive detection, characterization and monitoring of carcinoma. Many previous studies have shown that the number of CTCs has a significant relationship with the stage of cancer. However, CTC enrichment and detection remain notoriously difficult because they are extremely rare in the bloodstream. Herein, aided by a microfluidic device, an immunomagnetic separation system was applied to efficiently capture and in situ identify circulating tumor cells. Magnetic nanospheres (MNs) were modified with an anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody to fabricate immunomagnetic nanospheres (IMNs). IMNs were then loaded into the magnetic field controllable microfluidic chip to form uniform IMN patterns. The IMN patterns maintained good stability during the whole processes including enrichment, washing and identification. Apart from its simple manufacture process, the obtained microfluidic device was capable of capturing CTCs from the bloodstream with an efficiency higher than 94%. The captured cells could be directly visualized with an inverted fluorescence microscope in situ by immunocytochemistry (ICC) identification, which decreased cell loss effectively. Besides that, the CTCs could be recovered completely just by PBS washing after removal of the permanent magnets. It was observed that all the processes showed negligible influence on cell viability (viability up to 93%) and that the captured cells could be re-cultured for more than 5 passages after release without disassociating IMNs. In addition, the device was applied to clinical samples and almost all the samples from patients showed positive results, which suggests it could serve as a valuable tool for CTC enrichment and detection in the clinic.

Combination of dynamic magnetophoretic separation and stationary magnetic trap for highly sensitive and selective detection of Salmonella typhimurium in complex matrix.

Foodborne illnesses have always been a serious problem that threats public health, so it is necessary to develop a method that can detect the pathogens rapidly and sensitively. In this study, we designed a magnetic controlled microfluidic device which integrated the dynamic magnetophoretic separation and stationary magnetic trap together for sensitive and selective detection of Salmonella typhimurium (S. typhimurium). Coupled with immunomagnetic nanospheres (IMNs), this device could separate and enrich the target pathogens and realize the sensitive detection of target pathogens on chip. Based on the principle of sandwich immunoassays, the trapped target pathogens identified by streptavidin modified QDs (SA-QDs) were detected under an inverted fluorescence microscopy. A linear range was exhibited at the concentration from 1.0×10(4) to 1.0×10(6) colony-forming units/mL (CFU/mL), the limit of detection (LOD) was as low as 5.4×10(3) CFU/mL in milk (considering the sample volume, the absolute detection limit corresponded to 540C FU). Compared with the device with stationary magnetic trap alone, the integrated device enhanced anti-interference ability and increased detection sensitivity through dynamic magnetophoretic separation, and made the detection in complex samples more accurate. In addition, it had excellent specificity and good reproducibility. The developed system provides a rapid, sensitive and accurate approach to detect pathogens in practice samples.