Automatic Detection of Two Synovial Fluid Periprosthetic Joint Infection Biomarkers on an Integrated Microfluidic System.

Post-arthroplasty periprosthetic joint infection (PJI) is a serious ailment that can be difficult to diagnose. Herein, we developed a novel integrated microfluidic system (IMS) capable of detecting two common PJI biomarkers, alpha defensin human neutrophil peptide 1 (HNP-1) and C-reactive protein (CRP), from synovial fluid (SF). A magnetic bead-based one-aptamer-one-antibody assay was carried out automatically within 45 min on a single chip for simultaneous detection of both biomarkers at concentration ranges of 0.01-50 (HNP-1) and 1-100 (CRP) mg/L. It is the first report for utilizing these two biomarkers as targets to establish the new one-aptamer-one-antibody assay to detect PJI on-chip, and the aptamers demonstrated high specificity to their SF targets. As 20 clinical samples were correctly diagnosed with our IMS (verified by a common gold standard kit), it could serve as a promising tool for PJI diagnostics.

Integrated Microfluidic System for Cell-Free DNA Extraction from Plasma for Mutant Gene Detection and Quantification.

Ovarian cancer (OvCa) is among the most severe gynecologic cancers, yet individuals may be asymptomatic during its early stages. Routine, early screening for genetic abnormalities associated with OvCa could improve prognoses, and this can be achieved by detecting mutant genes in cell-free DNA (cfDNA). Herein, we developed an integrated microfluidic chip (IMC) that could extract cfDNA from plasma and automatically detect and quantify mutations in the OvCa biomarker BRCA1. The cfDNA extraction module relied on a vortex-type micromixer to mix cfDNA with magnetic beads surface-coated with cfDNA probes and could isolate 76% of molecules from a 200 µL plasma sample in 45 min. The cfDNA quantification module, which comprised a micropump that evenly distributed 4.5 µL of purified cfDNA into the on-chip, allele-specific quantitative polymerase chain reaction (qPCR) zones, was capable of quantifying mutant genes within 90 min. By automating the cfDNA extraction and qPCR processes, this IMC could be used for clinical screening for OvCa-associated mutations.

Isolation and Quantification of Methylated Cell-Free DNA in Plasma on an Integrated Microfluidic System.

Methylated cell-free DNA (cfDNA) has been deemed a promising biomarker for ovarian cancer (OvCa) prognosis and therapy selection. However, exploring the methylation profiles of tumor suppressor genes in cfDNA remains a challenge due to their extremely low concentrations and complicated protocols, as well as methodological constraints. In this study, an integrated microfluidic system was developed to automatically (1) capture methylated cfDNA in plasma by magnetic beads coated with the methyl-CpG-binding domain and (2) quantify the methylation level of tumor suppressor genes by on-chip quantitative polymerase chain reaction (qPCR). For capturing methylated cfDNA from a very small amount of plasma, samples along with beads were mixed in a new micromixer to enhance the capture rate. With a high capture rate (72%) and a limit of quantification of 0.1 pg/µL (3 orders of magnitude lower than that of the benchtop method), the compact system could detect the methylated cfDNA from only 20 µL of plasma sample in 2 h. Furthermore, the dynamic range, from 0.1 to 2000 pg/µL of methylated cfDNA, spans the physiological range in plasma, signifying that this device has great potential for personalized medicine in OvCa.

An integrated microfluidic platform featuring real-time reverse transcription loop-mediated isothermal amplification for detection of COVID-19.

An integrated microfluidic platform (IMP) utilizing real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) was developed here for detection and quantification of three genes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; i.e., coronavirus diseases 2019 (COVID-19)): RNA-dependent RNA polymerase, the envelope gene, and the nucleocapsid gene for molecular diagnosis. The IMP comprised a microfluidic chip, a temperature control module, a fluidic control module that collectively carried out viral lysis, RNA extraction, RT-LAMP, and the real-time detection within 90 min in an automatic format. A limit of detection of 5 × 103 copies/reaction for each gene was determined with three samples including synthesized RNAs, inactive viruses, and RNAs extracted from clinical samples; this compact platform could be a useful tool for COVID-19 diagnostics.

Design and Demonstration of Tunable Amplified Sensitivity of AlGaN/GaN High Electron Mobility Transistor (HEMT)-Based Biosensors in Human Serum.

We have developed a swift and simplistic protein immunoassay using aptamer functionalized AlGaN/GaN high electron mobility transistors (HEMTs). The unique design of the sensor facilitates protein detection in a physiological salt environment overcoming charge screening effects, without requiring sample preprocessing. This study reports a tunable and amplified sensitivity of solution-gated electric double layer (EDL) HEMT-based biosensors, which demonstrates significantly enhanced sensitivity by designing a smaller gap between the gate electrode and the detection, and by operating at higher gate voltage. Sensitivity is calculated by quantifying NT-proBNP, a clinical biomarker of heart failure, in buffer and untreated human serum samples. The biosensor depicts elevated sensitivity and high selectivity. Furthermore, detailed investigation of the amplified sensitivity in an increased ionic strength environment is conducted, and it is revealed that a high sensitivity of 80.54 mV/decade protein concentration can be achieved, which is much higher than that of previously reported FET biosensors. This sensor technology demonstrates immense potential in developing surface affinity sensors for clinical diagnostics.

Correction: Bacterial detection and identification from human synovial fluids on an integrated microfluidic system.

Correction for 'Bacterial detection and identification from human synovial fluids on an integrated microfluidic system' by Ting-Hang Liu et al., Analyst, 2019, 144, 1210-1222.

Optimization of an enzyme linked DNA aptamer assay for cardiac troponin I detection: synchronous multiple sample analysis on an integrated microfluidic platform.

In this study, an enzyme linked DNA aptamer based assay was optimized for human cardiac troponin I (cTnI) detection which is a prominent biomarker for acute myocardial infarction (AMI), on an integrated microfluidic platform. This platform allowed for the multiplex detection of six samples (5 µL per sample), and only 30 min were required for detection. First, cTnI-specific aptamers were surface-coated on magnetic beads. Bead-captured proteins were allowed to bind to a primary cTnI antibody and then to a secondary antibody labelled with horseradish peroxidase. Finally, chemiluminescence intensities were detected for quantification of cTnI. Purified proteins, serum from AMI patients and unknown serum samples were used to test the efficacy of the on-chip system. The limit of detection was measured to be only 12 ng L-1, and off-target effects from other proteins were minimal. This sensitive, cTnI-specific aptamer-based assay could consequently be used for reliable diagnosis of AMI.

Bacterial detection and identification from human synovial fluids on an integrated microfluidic system.

Periprosthetic joint infections (PJIs) are among the most severe complications emerging from prosthetic joint replacement surgeries. In order to possess a rapid means of diagnosing PJIs, an integrated microfluidic system was developed herein for detecting and identifying bacteria in human synovial fluid (HSF). The entire molecular diagnostic process, including (1) sample treatment, (2) bacterial isolation, (3) bacterial lysis, (4) nucleic acid amplification (via polymerase chain reaction (PCR)), and (5) optical detection, could be automated on a single chip. First, N-acetyl-l-cysteine was used to decrease the viscosity of HSF samples and consequently enhance bacterial isolation with vancomycin-coated nano-magnetic beads. Then, a universal 16S ribosomal ribonucleic acid PCR primer set and four species-specific primer sets were used for PCR-based detection and identification of four common bacteria previously associated with PJIs, including Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli, and Acinetobacter baumannii. With this approach, the limit of detection was as low as 100 colony forming units (CFUs) per milliliter (or 20 CFUs per reaction), which is suitable for clinical diagnostics and for making informed decisions regarding post-operative antibiotic administration. More importantly, bacterial detection and identification data could be acquired within 90 minutes, representing a significant improvement over traditional culture-based methods (3-7 days). The developed microfluidic system may therefore serve as a promising tool for rapid diagnosis of PJIs.

An automatic integrated microfluidic system for allergy microarray chips.

Billions of people suffer from allergies, though in many cases, the source allergen is unknown. If one knows which allergens to avoid, this would result in an improved quality of life. Since a rapid, high-throughput, automatic allergen detection method is of great need, an integrated system combining microfluidic techniques and microarray chips has been developed herein to automate the allergen detection process. The developed microfluidic system could automatically carry out the entire procedure such as reagent incubation, hybridization, transport, and washing without any intermediate step. The microarray chip could be easily detached from the microfluidic chip afterwards, enabling it to be read under a fluorescence scanner. The experimental results indicated that the developed microfluidic system can automatically perform all the incubation processes, including hybridization, reagent transportation, and washing. It is worth noting that active mixing has been applied in the present study which is different from our previous study using micro-channels for passive incubation. Comparable results to a conventional benchtop approach were obtained in ∼30% less time with ∼25% less samples/reagents. Similar results were also demonstrated while detecting immunoglobulin E samples. The developed system could therefore provide a rapid, reliable, and automated approach for detecting allergen-specific antibodies in human serum.

Determination of Cell Membrane Capacitance and Conductance via Optically Induced Electrokinetics.

Cell membrane capacitance and conductance are key pieces of intrinsic information correlated with the cellular dielectric parameters and morphology of the plasma membrane; these parameters have been used as electrophysiological biomarkers to characterize cellular phenotype and state, and they have many associated clinical applications. Here, we present our work on the non-invasive determination of cell membrane capacitance and conductance by an optically activated microfluidics chip. The model for determining the cell membrane capacitance and conductance was established by a single layer of the shell-core polarization model. Three-dimensional finite-element analyses of the positive and negative optically induced dielectrophoresis forces generated by the projected light arrays of spots were performed, thus providing a theoretical validation of the feasibility of this approach. Then, the crossover frequency spectra for four typical types of cells (Raji cells, MCF-7 cells, HEK293 cells, and K562 cells) were experimentally investigated by using a micro-vision based motion-tracking technique. The different responses of these cells to the positive and negative ODEP forces were studied under four different liquid conductivities by automatic observation and tracking of the cellular trajectory and texture during the cells' translation. The cell membrane capacitance and conductance were determined from the curve-fitted spectra, which were 11.1 ± 0.9 mF/m2 and 782 ± 32 S/m2, respectively, for Raji cells, 11.5 ± 0.8 mF/m2 and 114 ± 28 S/m2 for MCF-7 cells, 9.0 ± 0.9 mF/m2 and 187 ± 22 S/m2 for HEK293 cells, and 10.2 ± 0.7 mF/m2 and 879 ± 24 S/m2 for K562 cells. Furthermore, as an application of this technique, the membrane capacitances of MCF-7 cells treated with four different concentrations of drugs were acquired. This technique introduces a determination of cell membrane capacitance and conductance that yields statistically significant data while allowing information from individual cells to be obtained in a non-invasive manner.

Application of strong transverse magneto-optical Kerr effect on high sensitive surface plasmon grating sensors.

A high sensitive sensor is demonstrated by exploiting strong transverse magneto-optical Kerr effect on a ferromagnetic surface plasmon grating. The surface plasmon grating, made of a hybridized Au/Fe/Au layer, exhibits a very dispersive Kerr parameter variation near the surface plasmon polariton (SPP) wavelength via coherent scattering of the SPP on the grating structure. Interrogating this Kerr parameter can be utilized for detecting chemical or biological objects in a fluid medium. The experiment results show the minimal detectable mass concentration of sodium chloride in a saline solution is 4.27 × 10(-3) %, corresponding to a refractive index change of 7.60 × 10(-6) RIU. For an avidin-biotin interaction experiment, the sensitivity of avidin detection in PBS solution is 1.97 nM, which is limited by the index fluctuation of flowing media during measurement.

Size-dependent attenuation of TLR9 signaling by gold nanoparticles in macrophages.

Gold nanoparticles (GNPs), which are generally thought to be bio-inert and non-cytotoxic, have become one of the most ideal nanomaterials for medical applications. Once engulfed by phagocytes, the immunological effects of GNPs are still of concern and require detailed investigation. Therefore, this study explored the immunological significance of GNPs on TLR-mediated innate immunity in murine macrophages. GNP causes specific inhibition of TLR9 (CpG oligodeoxynucleotides; CpG-ODNs) signal in macrophages. The impaired CpG-ODN-induced TNF-α production is GNP concentration- and size-dependent in murine Raw264.7 cells: a GNP of 4 nm in size is more potent than a GNP of 11, 19, 35, or 45 nm in size. Consistent with cytokine inhibition, the CpG-ODN-induced phosphorylation of NF-κB and JNK as well as NF-κB activation are suppressed by GNPs. GNPs accumulate in lysosomes after phagocytosis and also increase TLR9-associated lysosomal cathepsin expression and activities, but this is irrelevant to TLR9 inhibition by GNPs in our studies. In addition, GNPs affected TLR9 translocation in response to CpG-ODNs and to phagosomes. Further exploring how GNPs inhibited TLR9 function, we found that GNPs could bind to high-mobility group box-1 (which is involved in the regulation of TLR9 signaling) inside the lysosomes. The current studies demonstrate that size-dependent inhibition of TLR9 function by GNP may be attributed to its binding to high-mobility group box-1.

An integrated microfluidic device utilizing vancomycin conjugated magnetic beads and nanogold-labeled specific nucleotide probes for rapid pathogen diagnosis.

A PCR-free assay for rapid pathogen diagnosis was implemented on an integrated microfluidic system in this study. Vancomycin-conjugated magnetic beads were used to capture multiple strains of bacteria and nucleotide probes labeled gold nanoparticles were used to specify and detect a specific strain by hybridization-induced color change. The assay was entirely automated within an integrated microfluidic device that was composed of suction-type micropumps, microvalves, microchannels, and microchambers that fabricated by microfluidic technology. Multiple strains of bacteria could be captured simultaneously by vancomycin-conjugated magnetic beads, with capturing efficiency exceeding 80%. Subsequently, sensitive and strain-specific detection against target bacteria could be achieved by using nanogold labeled specific nucleotide probes. The limit of detection of 10(2)CFU bacteria was achieved. Importantly, nucleic acid amplification was not involved in the diagnostic procedures; the entire analytic process required only 25min. The developed platform may provide a promising tool for rapid diagnosis of bacterial infections. FROM THE CLINICAL EDITOR: In this novel study, a PCR-free pathogen detection method is demonstrated. After vancomycin-conjugated magnetic beads captured bacteria, nucleotide probes-labeled gold nanoparticles were employed to specify and detect specific strains via hybridization-induced color change. Multiple strains of bacteria could be captured simultaneously with an efficiency exceeding 80%, enabling the detection of as low as 10(2) CFU of bacteria.

Magnetic nanoparticle-based immunoassay for rapid detection of influenza infections by using an integrated microfluidic system.

Magnetic manganese ferrite (MnFe2O4) nanoparticles with approximately 100nm in diameter were used to improve the performance of an immunoassay for detecting influenza infections. The synthesized nanoparticles were tested for long-term storage to confirm the stability of their thermal decomposition process. Then, an integrated microfluidic system was developed to perform the diagnosis process automatically, including virus purification and detection. To apply these nanoparticles for influenza diagnosis, a micromixer was optimized to reduce the dead volume within the microfluidic chip. Furthermore, the mixing index of the micromixer could achieve as high as 97% in 2seconds. The optical signals showed that this nanoparticle-based immunoassay with dynamic mixing could successfully achieve a detection limit of influenza as low as 0.007 HAU. When compared with the 4.5-µm magnetic beads, the optical signals of the MnFe2O4 nanoparticles were twice as sensitive. Furthermore, five clinical specimens were tested to verify the usability of the developed system. FROM THE CLINICAL EDITOR: In this study, magnetic manganese ferrite nanoparticles were used to improve the performance of a novel immunoassay for the rapid and efficient detection of influenza infections.

Nanoscale sorting of extracellular vesicles via optically-induced dielectrophoresis on an integrated microfluidic system.

We reported a microfluidic system for sorting of extracellular vesicles (EVs), which can house DNAs, RNAs, lipids, proteins, and metabolites that are important in intercellular communication. Their presence within bodily fluids has demonstrated potential in both clinical diagnostic and therapeutic applications. Furthermore, EVs exhibit distinct subtypes categorized by their sizes, each endowed with unique biophysical properties. Despite several existing techniques for EV isolation and purification, diminished purity and prolonged processing times still hamper clinical utility; comprehensive capture of EVs remains an ongoing pursuit. To address these challenges, we devised an innovative method for automated sorting of nano-scale EVs employing optically-induced dielectrophoresis on an integrated microfluidic chip. With this approach, EVs of three distinct size categories (small: 100-150 nm, medium-sized: 150-225 nm, and large: 225-350 nm) could be isolated at a purity of 86%. This new method has substantial potential in expediting EV research and diagnostics.

Advancing the Diagnosis of Periprosthetic Joint Infections: Integrated Microfluidic Platform for Alpha-Defensins-Specific Aptamer Selection and Its Analytical Applications.

Periprosthetic joint infections (PJIs) pose a significant challenge in orthopedic surgery, particularly total joint arthroplasty (TJA), due to the potential for implant failure and increased patient morbidity. Early and accurate detection of PJIs is crucial for timely intervention and better patient prognosis. Herein, we successfully screened a high-affinity aptamer targeting alpha-defensin complex human neutrophil protein 1-3 (HNP 1-3; potential PJI biomarkers in synovial fluid [SF]) for the first time using systematic evolution of ligands by exponential enrichment (SELEX) on an integrated microfluidic platform. The compact microfluidic device enabled efficient screening, with each round completed within <2 h, comprising five rounds of positive selection, two rounds of negative selection, and one round of competitive selection. A novel one-aptamer-one-antibody assay was further developed from the optimal aptamer screened, and it could accurately quantify HNP 1-3 in SF within 3 h with only ∼50 µL of SF. The assay demonstrated strong binding affinity and specificity for the target protein in SF. Thirteen PJI SF samples were accurately diagnosed and the assay was accurate over a wide dynamic range (0.32-100 mg/L). This study has showcased a rapid and accurate diagnostic tool for PJI detection, which should see widespread use in the clinic, holding promise for potential analytical applications in orthopedic surgery and improving patient care.

A self-driven, microfluidic, integrated-circuit biosensing chip for detecting four cardiovascular disease biomarkers.

Cardiovascular diseases (CVDs) claimed the lives of nearly 21 million people worldwide in 2021, accounting for 30% of global deaths. However, one in five CVD patients is unaware that they have the disease, emphasizing the need for accurate biomarker monitoring. Herein we developed an integrated microfluidic system (IMS) for rapid quantification of four CVD biomarkers, including N-terminal pro B-type natriuretic peptide (NT-proBNP), fibrinogen, cardiac troponin I (cTnI), and C-reactive protein (CRP)- via aptamer-coated interdigitated electrodes (IDE) with integrated circuits (IC) and a self-driven IMS for sample treatment. The device was composed of plasma filtration, metering, and fluidic delay modules, and the former could extract 45% of plasma from a 20-µL blood sample; the metering module could quantify 5 µL of plasma within 90 s. Subsequently, the plasma was transported to a detection chamber, where IC-based IDE sensors made measurements within 5 min. The entire 15-min process allowed us to evaluate biomarkers across a wide dynamic range: NT-proBNP (0.1-10,000 pg/mL), fibrinogen (50-1,000 mg/dL), cTnI (0.1-10,000 pg/mL), and CRP (0.5-9 mg/L). Given that spiked blood samples were measured with reasonable accuracy (>80%), the IMS could see utility in CVD risk assessment and personalized medicine.

An integrated microfluidic system for automatic detection of cholangiocarcinoma cells from bile.

Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies (i.e. blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.

A microfluidic immunomagnetic bead-based system for the rapid detection of influenza infections: from purified virus particles to clinical specimens.

Seasonal and novel influenza infections have the potential to cause worldwide pandemics. In order to properly treat infected patients and to limit its spread, a rapid, accurate and automatic influenza diagnostic tool needs to be developed. This study therefore presents a new integrated microfluidic system for the rapid detection of influenza infections. It integrated a suction-type, pneumatic-driven microfluidic control module, a magnetic bead-based fluorescent immunoassay (FIA) and an end-point optical detection module. This new system can successfully distinguish between influenza A and B using a single chip test within 15 min automatically, which is faster than existing devices. By utilizing the micromixers to thoroughly wash out the sputum-like mucus, this microfluidic system could be used for the diagnosis of clinical specimens and reduced the required sample volume to 40 µL. Furthermore, the results of diagnostic assays from 86 patient specimens have demonstrated that this system has 84.8 % sensitivity and 75.0 % specificity. This developed system may provide a powerful platform for the fast screening of influenza infections.

An integrated microfluidic platform for rapid tumor cell isolation, counting and molecular diagnosis.

Ovarian cancer is the second most common of the gynecological cancers in Taiwan. It is challenging to diagnose at an early stage when proper treatment is the most effective. It is well recognized that the detection of tumor cells (TCs) is critical for determining cancer growth stages and may provide important information for accurate diagnosis and even prognosis. In this study, a new microfluidic platform integrated with a moving-wall micro-incubator, a micro flow cytometer and a molecular diagnosis module performed automated identification of ovarian cancer cells. By efficiently mixing the cells and immunomagnetic beads coated with specific antibodies, the target TCs were successfully isolated from the clinical samples. Then counting of the target cells was achieved by a combination of the micro flow cytometer and an optical detection module and showed a counting accuracy as high as 92.5 %. Finally, cancer-associated genes were amplified and detected by the downstream molecular diagnosis module. The fluorescence intensity of specific genes (CD24 and HE4) associated with ovarian cancer was amplified by the molecular diagnosis module and the results were comparable to traditional slab-gel electrophoresis analysis, with a limit of detection around 10 TCs. This integrated microfluidic platform realized the concept of a "lab-on-a-chip" and had advantages which included automation, disposability, lower cost and rapid diagnosis and, therefore, may provide a promising approach for the fast and accurate detection of cancer cells.