A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases.

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases.

Immuno-PET Imaging of TNF-α in Colitis Using 89Zr-DFO-infliximab.

Tumor necrosis factor-alpha (TNF-α) neutralization has become increasingly important in the treatment of inflammatory bowel diseases (IBD). A series of monoclonal antibodies were approved in the clinic for anti-TNF-α therapy. However, a comprehensive assessment of TNF-α levels throughout the colon, which facilitates the diagnosis of IBD and predicts anti-TNF-α efficacy, remains challenging. Here, we radiolabeled infliximab with long-lived radionuclides 89Zr for immuno-positron emission tomography (PET) imaging of TNF-α in vivo. The increased TNF-α level was detected in the inflammatory colon of the dextran sodium sulfate-induced colitis mice. The immuno-PET imaging of 89Zr-desferrioxamine-infliximab reveals a high uptake (7.1 ± 0.3%ID/g) in the inflammatory colon, which is significantly higher than in the healthy control and blocked groups. The colon-to-muscle ratio reached more than 10 and was maintained at a high level for 10 h after injection. The ex vivo biodistribution study also verified the superior uptake in the inflammatory colon. This study provides an in vivo immune-PET approach to molecular imaging of the pro-inflammatory cytokine TNF-α. It is promising in diagnosing and predicting efficacy in both IBD and other autoimmune diseases.

Simple and rapid separation of Haematococcus pluvialis and ciliate based on the dean-coupled inertial microfluidics.

Astaxanthin with high antioxidant activity is of great practical value and Haematococcus pluvialis is recognized as the best natural astaxanthin producer. The yield of Haematococcus pluvialis was often affected by the ciliate during its production, however, the use of biochemical pesticides might have a great impact on Haematococcus pluvialis. Therefore, a simple microfluidic chip with the spiral microchannel was developed for continuous-flow physical separation of ∼10 µm ciliate from ∼30 µm Haematococcus pluvialis since their different sizes resulted in different equilibrium positions in the channel due to the Dean-coupled inertial migration. First, a spiral microchannel with a width of 700 µm and a height of 130 µm in the microfluidic chip was developed using three-dimensional printing and verified to completely separate polystyrene particles of 10 µm from those of 30 µm. Then, this microfluidic chip was used to separate the actual sample, and experimental results showed that ∼80% of ciliate was continuously separated from Haematococcus pluvialis at a flow rate of 2.8 ml/min. More importantly, no additional biochemical reagents were used and the activity of Haematococcus pluvialis was not affected. This microfluidic chip featured with simple design, automatic operation, and small size is promising for purification and breeding of Haematococcus pluvialis.

Recent Advances on Bioaerosol Collection and Detection in Microfluidic Chips.

Bioaerosols containing pathogenic microorganisms have posed a great threat to human and animal health. Effective monitoring of bioaerosols containing pathogenic viruses and bacteria is of great significance to prevent and control infectious diseases. This Feature summarizes recent advances on bioaerosol collection and detection based on microfluidic chips. Besides, the challenges and trends for bioaerosol collection and detection were also discussed.

A colorimetric immunosensor for determination of foodborne bacteria using rotating immunomagnetic separation, gold nanorod indication, and click chemistry amplification.

A colorimetric immunosensor was developed for the determination of Salmonella Typhimurium using rotating magnetic separation, gold nanorod (GNR) indication, and click chemistry amplification. The target bacteria were first separated from large-volume sample using a rotating magnetic field and a small amount (50 µg) of immunomagnetic nanoparticles (MNPs), resulting in the forming of magnetic bacteria. Then, the magnetic bacteria were conjugated with catalase (CAT)-labeled antibodies, which were synthesized using trans-cyclooctene/1,2,4,5-tetrazine click chemistry reaction, resulting in the forming of enzymatic bacteria. Then the CATs on the enzymatic bacteria were used to decompose an excessive amount of hydrogen peroxide (H2O2), the remaining H2O2 was mixed with horseradish peroxidase to etch the GNRs, resulting in color change and absorbance peak shift of the GNRs. Finally, the peak shift was measured and analyzed for the quantitative determination of target bacteria. This immunosensor was able to detect Salmonella Typhimurium with a linear range of 101-105 CFU mL-1 in 3 h with a low detection limit of 35 CFU mL-1. The mean recovery for Salmonella Typhimurium in spiked chicken samples was 109%. Graphical abstractSchematic representation of a colorimetric immunosensor for the determination of Salmonella Typhimurium as low as 35 CFU mL-1 using rotating magnetic separation of Salmonella from a large-volume sample, click chemistry reaction of catalase with antibodies for signal amplification, and HRP-mediated gold nanorod etching for result indication.

A Rapid and Sensitive Salmonella Biosensor Based on Viscoelastic Inertial Microfluidics.

Salmonella is a main cause of foodborne illnesses and rapid screening of Salmonella is the key to prevent Salmonella outbreaks, however available detection methods either require a long time, or need complex pretreatment, or have low sensitivity. In this study, a microfluidic biosensor was developed for Salmonella detection using viscoelastic inertial microfluidics for separating magnetic bacteria from unbound magnetic nanoparticles (MNPs) and enzyme catalytic colorimetry for amplifying biological signals. The polyclonal antibodies and horseradish peroxidase (HRP) modified MNPs were first used to specifically capture Salmonella to form magnetic HRP-bacteria. Both magnetic HRP-bacteria and unbound MNPs were magnetically separated from background and resuspended in viscoelastic polyvinylpyrrolidone solution as sample flow. When sample flow was injected with polyvinylpyrrolidone sheath flow into a T-shaped microchannel, larger-sized magnetic HRP-bacteria could penetrate the sample flow, however smaller-sized MNPs remained in the sample flow due to weaker inertial lift force and elastic lift force, resulting in continuous-flow separation of magnetic HRP-bacteria. Finally, magnetic HRP-bacteria were collected and concentrated to catalyze tetramethyl benzidine, and absorbance was measured to determine the bacteria. This biosensor was able to detect Salmonella as low as 30 CFU/mL in 1 h and featured the advantages of shorter time due to a one-step immunoreaction, easier extension due to only one antibody and one label, and lower cost due to less expensive materials.

A general-purpose signal processing algorithm for biological profiles using only first-order derivative information.

BACKGROUND: Automatic signal-feature extraction algorithms are crucial for profile processing in bioinformatics. Both baseline drift and noise seriously affect the position and peak area of signals. An efficient algorithm named the derivative passing accumulation (DPA) method for simultaneous baseline correction and signal extraction is presented in this article. It is an efficient method using only the first-order derivatives which are obtained through taking the simple differences. RESULTS: We developed a new signal feature extracting procedure. The vector representing the discrete first-order derivative was divided into negative and positive parts and then accumulated to build a signal descriptor. The signals and background fluctuations are easily separated according to this descriptor via thresholding. In addition, the signal peaks are simultaneously located by checking the corresponding intervals in the descriptor. Therefore, the eternal issues of parsing the 1-dimensional output of detectors in biological instruments are solved together. Thereby, the baseline is corrected, and the signal peaks are extracted. CONCLUSIONS: We have introduced a new method for signal peak picking, where baseline computation and peak identification are performed jointly. The testing results of both authentic and artificially synthesized data illustrate that the new method is powerful, and it could be a better choice for practical processing.

A microfluidic immunosensor for visual detection of foodborne bacteria using immunomagnetic separation, enzymatic catalysis and distance indication.

A disposable visual microfluidic immunosensor is described for the determination of foodborne pathogens using immunomagnetic separation, enzymatic catalysis and distance indication. Specifically, a sensor was designed to detect Salmonella typhimurium as a model pathogen. Magnetic nanoparticles (MNPs) were modified with the anti-Salmonella monoclonal antibodies and then used to enrich S. typhimurium from the sample. This is followed by conjugation to polystyrene microspheres modified with anti-Salmonella polyclonal antibodies and catalase to form the MNP-bacteria-polystyrene-catalase sandwich. The catalase on the complexes catalyzes the decomposition of hydrogen peroxide to produce oxygen after passing a micromixer. The generated oxygen gas increases the pressure in the chip and pushes the indicating red dye solution to travel along the channel towards the unsealed outlet. The travel distance of the red dye can be visually read and related to the amount of S. typhimurium using the calibration scale. The sensor can detect as low as 150 CFU·mL-1 within 2 h. Graphical abstractSchematic representation of the distance-based microfluidic immunosensor for visual detection of foodborne bacteria using immunomagnetic nanoparticles for bacteria separation, catalase for decomposition of hydrogen peroxide to form oxygen which causes a pressure increase, and red dyed particles movement for distance indication.

Tunable, Sheathless Focusing of Diamagnetic Particles in Ferrofluid Microflows with a Single Set of Overhead Permanent Magnets.

There has been increasing interest in the use of magnetic fluids to manipulate diamagnetic particles in microfluidic devices. Current methods for diamagnetic-particle focusing in magnetic fluids require either a pair of repulsive magnets or a diamagnetic sheath flow. We demonstrate herein a tunable, sheathless focusing of diamagnetic particles in a microchannel ferrofluid flow with a single set of overhead permanent magnets. Particles are focused into a single stream near the bottom wall of a straight rectangular microchannel, where a magnetic-field minimum is formed as a result of the magnetization of the ferrofluid. This focusing can be readily switched off and on by removing and replacing the permanent magnets. More importantly, the particle-focusing position can be tuned by shifting the magnets with respect to the microchannel. We perform a systematic experimental study of the parametric effects of the fluid-particle-channel system on diamagnetic-particle focusing in terms of a defined particle-focusing effectiveness.

Rapid and sensitive detection of Escherichia coli O157:H7 using coaxial channel-based DNA extraction and microfluidic PCR.

In this study, a rapid and sensitive method for detection of Escherichia coli O157:H7 using the coaxial channel-based DNA extraction and the microfluidic PCR was proposed and verified. The magnetic silica beads were first pumped into the coaxial channel, which was captured in the coaxial channel more uniformly by applying the multiring high-gradient magnetic field. After the E. coli O157:H7 cells were lysed with the lysis buffer to release the DNA, the improved coaxial channel was used to efficiently extract the DNA, followed by washing with ethanol to remove the residual proteins and eluting with a small volume of deionized water to obtain the purified and concentrated DNA. Finally, the obtained DNA was amplified and determined using the microfluidic PCR. This proposed bacteria detection method was able to detect E. coli O157:H7 as low as 12 cfu/mL when the large volume (10 mL) of bacterial sample was used, and the recovery of E. coli O157:H7 in the spiked milk samples ranged from 97.4 to 100.6%. This proposed bacteria detection method has shown great potential to detect lower concentration of E. coli O157:H7 from larger volumes of sample.

Simultaneous Separation and Washing of Nonmagnetic Particles in an Inertial Ferrofluid/Water Coflow.

Magnetic fluids (e.g., paramagnetic solutions and ferrofluids) have been increasingly used for label-free separation of nonmagnetic particles in microfluidic devices. Their biocompatibility, however, becomes a concern in high-throughput or large-volume applications. One way to potentially resolve this issue is resuspending the particles that are separated in a magnetic fluid immediately into a biocompatible buffer. We demonstrate herein the proof-of-principle of the first integration of negative magnetophoresis and inertial focusing for a simultaneous separation and washing of nonmagnetic particles in coflowing ferrofluid and water streams. The two operations take place in parallel in a simple T-shaped rectangular microchannel with a nearby permanent magnet. We find that the larger and smaller particles' exiting positions (and hence their separation distance) in the sheath water and ferrofluid suspension, respectively, vary with the total flow rate or the flow rate ratio between the two streams.

Three-dimensional printed magnetophoretic system for the continuous flow separation of avian influenza H5N1 viruses.

As a result of the low concentration of avian influenza viruses in samples for routine screening, the separation and concentration of these viruses are vital for their sensitive detection. We present a novel three-dimensional printed magnetophoretic system for the continuous flow separation of the viruses using aptamer-modified magnetic nanoparticles, a magnetophoretic chip, a magnetic field, and a fluidic controller. The magnetic field was designed based on finite element magnetic simulation and developed using neodymium magnets with a maximum intensity of 0.65 T and a gradient of 32 T/m for dragging the nanoparticle-virus complexes. The magnetophoretic chip was designed by SOLIDWORKS and fabricated by a three-dimensional printer with a magnetophoretic channel for the continuous flow separation of the viruses using phosphate-buffered saline as carrier flow. The fluidic controller was developed using a microcontroller and peristaltic pumps to inject the carrier flow and the viruses. The trajectory of the virus-nanoparticle complexes was simulated using COMSOL for optimization of the carrier flow and the magnetic field, respectively. The results showed that the H5N1 viruses could be captured, separated, and concentrated using the proposed magnetophoretic system with the separation efficiency up to 88% in a continuous flow separation time of 2 min for a sample volume of 200 µL.

A Review on Magnetophoretic Immunoseparation.

Magnetophoresis is a motion of a magnetic or magnetizable particle induced by an inhomogeneous magnetic field in a fluid. Magnetophoretic immunoseparation, using micro- or nano-sized magnetic particles often modified by monoclonal or polyclonal antibodies for specific separation of biological or chemical targets, has shown a great potential in continuous-flow separation of cells and bacteria in clinical and biomedical fields. In this paper, the basic knowledge, key design considerations and recent developments on magnetophoretic immuno-separation of biological targets were reviewed.

A Magnetic Nanoparticle Based Nucleic Acid Isolation and Purification Instrument for DNA Extraction of Escherichia Coli O157: H7.

The objective of this study was to evaluate the performance of a nucleic acid isolation and purification instrument using Escherichia coli O157:H7 as the model. The instrument was developed with magnetic nanoparticles for efficiently capturing nucleic acids and an intelligent mechanical unit for automatically performing the whole nucleic acid extraction process. A commercial DNA extraction kit from Huier Nano Company was used as reference. Nucleic acids in 1 ml of E. coli O157: H7 at a concentration of 5 x 10(8) CFU/mL were extracted by using this instrument and the kit in parallel and then detected by an ultraviolet spectrophotometer to obtain A260 values and A260/A280 values for the determination of the extracted DNA's quantity and purity, respectively. The A260 values for the instrument and the kit were 0.78 and 0.61, respectively, and the A260/A280 values were 1.98 and 1.93. The coefficient of variations of these parallel tests ranged from 10.5% to 16.7%. The results indicated that this nucleic acid isolation and purification instrument could extract a comparable level of nucleic acid within 50 min compared to the commercial DNA extraction kit.

Exploiting enzyme catalysis in ultra-low ion strength media for impedance biosensing of avian influenza virus using a bare interdigitated electrode.

Enzyme catalysis is broadly used in various fields but generally applied in media with high ion strength. Here, we propose the exploitation of enzymatic catalysis in ultra-low ion strength media to induce ion strength increase for developing a novel impedance biosensing method. Avian influenza virus H5N1, a serious worldwide threat to poultry and human health, was adopted as the analyte. Magnetic beads were modified with H5N1-specific aptamer to capture the H5N1 virus. This was followed by binding concanavalin A (ConA), glucose oxidase (GOx), and Au nanoparticles (AuNPs) to create bionanocomposites through a ConA-glycan interaction. The yielded sandwich complex was transferred to a glucose solution to trigger an enzymatic reaction to produce gluconic acid, which ionized to increase the ion strength of the solution, thus decreasing the impedance on a screen-printed interdigitated array electrode. This method took advantages of the high efficiency of enzymatic catalysis and the high susceptibility of electrochemical impedance on the ion strength and endowed the biosensor with high sensitivity and a detection limit of 8 × 10(-4) HAU in 200 µL sample, which was magnitudes lower than that of some analogues based on biosensing methods. Furthermore, the proposed method required only a bare electrode for measurements of ion strength change and had negligible change on the surficial properties of the electrode, though some modification of magnetic beads/Au nanoparticles and the construction of a sandwich complex were still needed. This helped to avoid the drawbacks of commonly used electrode immobilization methods. The merit for this method makes it highly useful and promising for applications. The proposed method may create new possibilities in the broad and well-developed enzymatic catalysis fields and find applications in developing sensitive, rapid, low-cost, and easy-to-operate biosensing and biocatalysis devices.

Multiplex nanozymatic biosensing of Salmonella on a finger-actuated microfluidic chip.

A colorimetric biosensor was elaboratively designed for fast, sensitive and multiplex bacterial detection on a single microfluidic chip using immune magnetic nanobeads for specific bacterial separation, immune gold@platinum palladium nanoparticles for specific bacterial labeling, a finger-actuated mixer for efficient immunoreaction and two coaxial rotatable magnetic fields for magnetic nanobead capture (outer one) and magnet-actuated valve control (inner one). First, preloaded bacteria, nanobeads and nanozymes were mixed through a finger actuator to form nanobead-bacteria-nanozyme conjugates, which were captured by the outer magnetic field. After the inner magnetic field was rotated to successively wash the conjugates and push the H2O2-TMB substrate for resuspending these conjugates, colorless TMB was catalyzed into blue TMBox products, followed by color analysis using ImageJ software for bacterial determination. This simple biosensor enabled multiplex Salmonella detection as low as 9 CFU per sample in 45 min.

A three-in-one hybrid nanozyme for sensitive colorimetric biosensing of pathogens.

Pathogen screening is an important step in preventing foodborne diseases. In this study, a hybrid nanozyme, metal organic framework decorated with palladium (Pd) and platinum (Pt) (MIL-88@Pd/Pt), was innovatively synthesized and used with immune magnetic nanobeads (MNBs) for sensitive biosensing of Salmonella. First, immune MIL-88@Pd/Pt nanozymes and immune MNBs were mixed with target pathogens in a large-volume sample, resulting in effective isolation and specific label of target pathogens to form nanobead-Salmonella-nanozyme conjugates. Then, these conjugates were used to catalyze H2O2-TMB, and its color was changed from colorless to blue. Finally, catalysate absorption was measured to determine pathogen concentration. This colorimetric immunoassay could determine Salmonella typhimurium from 4 × 101 to 4 × 105 CFU/mL in 60 min with a detection limit of 32 CFU/mL.

Magnetic nanobead chain-assisted real-time impedance monitoring using PCB interdigitated electrode for Salmonella detection.

Pathogen testing is effective to prevent food poisoning. Here, an electrochemical biosensor was explored for Salmonella detection by combining magnetic grid based bacterial separation with enzymatic catalysis based signal amplification on a PCB interdigitated electrode in a microfluidic chip. First, immune magnetic nanobeads, target bacteria, and immune polystyrene microspheres decorated with glucose oxidase were sufficiently mixed to form nanobead-bacteria-microsphere sandwich conjugates. Then, these conjugates were injected into the chip to form conjugate chains right over the electrode under an iron grid enhanced magnetic field. After non-conductive glucose was injected and catalyzed by glucose oxidase on the conjugate chains, conductive glucose acid and non-conductive hydrogen peroxide were continuously produced and rapidly diffused from the conjugate chains to the electrode. Finally, the impedance change was real-timely monitored and used to determine the bacterial amount. This sensor enabled detection of 50 CFU/mL Salmonella typhimurium in 1 h.

Cost-Efficient Micro-Well Array-Based Colorimetric Antibiotic Susceptibility Testing (MacAST) for Bacteria from Culture or Community.

Rapid and cost-efficient antibiotic susceptibility testing (AST) is key to timely prescription-oriented diagnosis and precision treatment. However, current AST methods have limitations in throughput or cost effectiveness, and are impractical for microbial communities. Here, we developed a high-throughput micro-well array-based colorimetric AST (macAST) system equipped with a self-developed smartphone application that could efficiently test sixteen combinations of bacteria strains and antibiotics, achieving comparable AST results based on resazurin metabolism assay. For community samples, we integrated immunomagnetic separation into the macAST (imacAST) system to specifically enrich the target cells before testing, which shortened bacterial isolation time from days to only 45 min and achieved AST of the target bacteria with a low concentration (~103 CFU/mL). This proof-of-concept study developed a high-throughput AST system with an at least ten-fold reduction in cost compared with a system equipped with a microscope or Raman spectrum. Based on colorimetric readout, the antimicrobial susceptibility of the bacteria from microbial communities can be delivered within 6 h, compared to days being required based on standard procedures, bypassing the need for precise instrumentation in therapy to combat bacterial antibiotic resistance in resource-limited settings.

A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection.

BACKGROUND: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. METHODS: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. RESULTS: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 102 CFU/mL Salmonella Typhimurium was over 2000 RFU, while 104 CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). CONCLUSIONS: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.