Label-free microfluidic cell sorting and detection for rapid blood analysis.

Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics.

Power-free microfluidic biosensing of Salmonella with slide multivalve and disposable syringe.

In this study, a power-free biosensor was presented to detect Salmonella typhimurium on a microfluidic chip using a slide multivalve for channel selection and a disposable syringe for fluidic transfer. First, bacterial sample with immunomagnetic nanoparticles (IMNPs) and glucose oxidase (GOx) modified immune polystyrene nanoparticles (IPNPs), washing buffer, glucose, and peroxide test strip (PTS) were preloaded in their respective chambers at the periphery of chip. After the slide multivalve was selected to connect sample chamber with common separation chamber, which was connected with a syringe, the mixture of Salmonella, IMNPs and IPNPs was back and forth moved through 3D Tesla-structure micromixer using the syringe, resulting in the formation of IMNP-Salmonella-IPNP complexes, which were captured in the separation chamber using a magnet. Then, two washing chambers were selectively connected respectively to remove sample background and excessive IPNPs, and glucose chamber was connected, allowing the GOx to catalyze glucose to produce hydrogen peroxide in the separation chamber. Finally, PTS chamber was connected and the catalysate was transferred from the separation chamber to the PTS chamber, leading to the color change of PTS, followed by using smartphone App to collect and analyze the image of PTS for bacterial determination. The simple biosensor enabled simple detection of Salmonella as few as 130 CFU/mL within 60 min and is promising for practical applications in the resource-limited regions due to its low cost, simple operation, and small size.

Microfluidic Colorimetric Biosensors Based on MnO2 Nanozymes and Convergence-Divergence Spiral Micromixers for Rapid and Sensitive Detection of Salmonella.

In-field screening of foodborne pathogens plays an important role in ensuring food safety. Thus, a microfluidic biosensor was developed for rapid and sensitive detection of Salmonella using manganese dioxide nanoflowers (MnO2 NFs) for amplifying the biological signal, a microfluidic chip with a convergence-divergence spiral micromixer for performing automatic operations, and a smartphone app with a saturation calculation algorithm for processing the image. First, immune magnetic nanoparticles (MNPs), the sample, and immune MnO2 NFs were fully mixed and sufficiently incubated in the spiral micromixer to form MNP-bacteria-MnO2 sandwich complexes, which were magnetically captured in a separation chamber in the microfluidic chip. Then, a 3,3',5,5'-tetramethylbenzidine (TMB) substrate was injected and catalyzed by a MnO2 NF nanomimetic enzyme on the complexes, resulting in the production of yellow catalysate. Finally, the catalysate was transferred into a detection chamber and its image was measured and processed using the smartphone app to determine the number of bacteria. This biosensor was able to detect Salmonella from 4.4 × 101 to 4.4 × 106 CFU/mL in 45 min with a detection limit of 44 CFU/mL, and has the potential to provide a promising platform for on-site detection of foodborne bacteria.

The hospital management practices in Chinese county hospitals and its association with quality of care, efficiency and finance.

BACKGROUND: County hospitals as the backbone of the China's healthcare system are providing services for over 70% of the total population. However, the hospital management practice (HMP) and its links with quality of care, efficiency and finance in these hospitals are unknown. METHODS: We did two cross-sectional surveys of HMP in 2013 and 2015 among 101 county hospitals across rural China. Three managing roles (hospital director, director of medical affairs office and director of cardiology) and a cardiologist were invited to the surveys. A novel HMP rating scale, with 100 as full score, was used to measure the HMP in 17 indicators under four dimensions (target, operation, performance, and talent management) for each hospital. We analyzed the association of HMP score with variables on quality of care, efficiency and finance using linear mixed models with and without adjustment for potential confounders. FINDINGS: A total of 95 hospitals participated in at least one survey and were included in the analysis. The overall mean HMP score varied dramatically across the hospitals and 84% of them scored less than 60. The dimension mean HMP score was 38.6 (target), 56.4 (operation), 53.2 (performance) and 55.7 (talent), respectively. The pattern of indicator mean HMP score, however, was almost identical between hospitals with high and low overall HMP score, showing the same 'strength' (staff satisfaction, staff performance appraisal, 'hard wares', patient-centered services, etc.) and 'weakness' (target balance, target setting, continuous quality improvement, penalties on staff with dissatisfied performance, etc.). The associations of overall mean HMP score with quality of care and efficiency variables and cost per hospitalization was not statistically significant. The statistical significance in the association with hospital annual total income disappeared after adjusting for region, teaching status, number of competitors, number of staff and number of beds in use. CONCLUSION: The HMP in Chinese county hospitals scores low in general and was not significantly associated with hospital care quality, efficiency and finance. The current healthcare reform in China should address the micro level issues in hospital management practices.

An impedance biosensor based on magnetic nanobead net and MnO2 nanoflowers for rapid and sensitive detection of foodborne bacteria.

Screening of pathogenic bacteria in foods is an effective way to prevent foodborne diseases. In this study, an impedance biosensor was developed for rapid and sensitive detection of Salmonella typhimurium using multiple magnetic nanobead (MNB) nets in a ring channel for continuous-flow separation of target bacteria from 10 mL of sample, manganese dioxide nanoflowers (MnO2 NFs) for efficient amplification of biological signal, and an interdigitated microelectrode for sensitive measurement of impedance change. First, the MNBs modified with capture antibodies were vortically injected from outer periphery of this ring channel to form multiple ring MNB nets at specific locations with high gradient magnetic fields. Then, the bacterial sample was continuous-flow injected, resulting in specific capture of target bacteria onto the nets, and the MnO2 NFs modified with detection antibodies were injected to form MNB-bacteria-MnO2 NF complexes. After the complexes were washed with deionized water to remove excessive nanoflowers and residual ions, H2O2 with poor conductivity was injected to reduce MnO2 NFs to conductive Mn2+ at neutral medium, leading to impedance decrease. Finally, impedance change was measured using the microelectrode for quantitative determination of Salmonella. This biosensor was able to separate ~60% of Salmonella from 10 mL of bacterial sample and detect Salmonella with a linear range of 3.0 × 101 to 3.0 × 106 CFU/mL in 1.5 h with lower detection limit of 19 CFU/mL. This biosensor might be further improved with higher sensitivity using a larger volume (100 mL or more) for routine screening of foodborne bacteria without bacterial pre-culture.

Combining impedance biosensor with immunomagnetic separation for rapid screening of Salmonella in poultry supply chains.

Salmonella screening is a key to ensure food safety in poultry supply chains. Currently available Salmonella detection methods including culture, polymerase chain reaction and enzyme-linked immuno-sorbent assay could not achieve rapid, sensitive, and in-field detection. In this study, different strategies for separation and detection of Salmonella were proposed, compared, and improved based on our previous studies on immunomagnetic separation and impedance biosensor. First, the coaxial capillary for immunomagnetic separation of target bacteria was improved with less contamination, and 3 strategies based on the improved capillary and immunomagnetic nanoparticles were compared to separate the target bacteria from sample and form the magnetic bacteria. The experimental results showed that the strategy of capture in tube and separation in capillary was the most suitable with separation efficiency of approximately 88%. Then, the immune gold nanoparticles coated with urease were used to label the magnetic bacteria, resulting in the formation of enzymatic bacteria, which were injected into the capillary. After the urea was catalyzed by the urease on the enzymatic bacteria in the capillary, different electrodes were compared to measure the impedance of the catalysate and the screen-printed electrode with higher sensitivity and better stability was the most suitable. This impedance biosensor-based bacterial detection strategy was able to detect Salmonella as low as 102 CFU/mL in 2 h without complex operations. Compared to the gold standard culture method for practical screening of Salmonella in poultry supply chains, this proposed strategy had an accuracy of approximately 90% for 75 real poultry samples.

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.

Exploring Protein-Inorganic Hybrid Nanoflowers and Immune Magnetic Nanobeads to Detect Salmonella Typhimurium.

Early screening of pathogenic bacteria is key to preventing and controlling outbreaks of foodborne diseases. In this study, protein-inorganic hybrid nanoflowers were synthesized for signal amplification and used with a calcium ion selective electrode (Ca-ISE) to establish a new enzyme-free assay for rapid and sensitive detection of Salmonella. Calcium hydrophosphate crystals were first conjugated with polyclonal antibodies against Salmonella to synthesize immune calcium nanoflowers (CaNFs), and streptavidin modified magnetic nanobeads (MNBs) were conjugated with biotinylated monoclonal antibodies against Salmonella to form immune MNBs. After target bacteria were separated using immune MNBs to form magnetic bacteria, immune CaNFs were conjugated with magnetic bacteria to form nanoflower conjugated bacteria. Then, hydrogen chloride was used to release calcium ions from nanoflower conjugated bacteria. After magnetic separation, the supernatant was finally injected as a continuous-flow to fluidic chip with Ca-ISE for specific detection of calcium ions. The supernatant's potential had a good linear relationship with bacteria concentration, and this assay was able to detect the S. Typhimurium cells as low as 28 colony forming units/mL within two hours. The mean recovery of target bacteria in spiked chicken samples was 95.0%. This proposed assay shows the potential for rapid, sensitive, and on-line detection of foodborne pathogens.

An enzyme-free biosensor for sensitive detection of Salmonella using curcumin as signal reporter and click chemistry for signal amplification.

In this study, an enzyme-free biosensor was developed for sensitive and specific detection of Salmonella typhimurium (S. typhimurium) using curcumin (CUR) as signal reporter and 1,2,4,5-tetrazine (Tz)-trans-cyclooctene (TCO) click chemistry for signal amplification. METHODS: Nanoparticles composed of CUR and bovine serum albumin (BSA) were formulated and reacted with Tz and TCO to form Tz-TCO-CUR conjugates through Tz-TCO click chemistry. Then, the Tz-TCO-CUR conjugates were functionalized with polyclonal antibodies (pAbs) against S. typhimurium to form CUR-TCO-Tz-pAb conjugates. Magnetic nanoparticles (MNPs) conjugated with monoclonal antibodies (mAbs) against S. typhimurium through streptavidin-biotin binding were used to specifically and efficiently separate S. typhimurium from the background by magnetic separation. CUR-TCO-Tz-pAb conjugates were reacted with the magnetic bacteria to form CUR-Tz-TCO bacteria. Finally, CUR was released quickly from the CUR-Tz-TCO bacteria in the presence of NaOH, and the color change was measured at the characteristic wavelength of 468 nm for bacteria quantification. RESULTS: A linear relationship between absorbance at 468 nm and concentration of S. typhimurium from 102 to 106 CFU/mL was found. The lower detection limit was calculated to be as low as 50 CFU/mL and the mean recovery was 107.47% for S. typhimurium in spiked chicken samples. CONCLUSION: This biosensor has the potential for practical applications in the detection of foodborne pathogens.