Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

The expression of programmed death-ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput droplet-based extracellular vesicle analysis (DEVA) assay for ultrasensitive quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs. We achieve a performance that significantly surpasses conventional approaches, demonstrating 360× enhancement in the limit of detection (LOD) and a 750× improvement in the limit of quantitation (LOQ) compared to conventional plate enzyme-linked immunoassay (ELISA). Underlying this performance is DEVA's high throughput analysis of individual EVs one at a time and the high specificity to targeted EVs versus background. We achieve a 0.006% false positive rate per droplet by leveraging avidity effects that arise from EVs having multiple copies of their target ligands on their surface. We use parallelized optofluidics to rapidly process 10 million droplets per minute, ∼100× greater than conventional approaches. A validation study on a cohort of 14 patients with melanoma confirms DEVA's ability to match conventional ELISA measurements with reduced plasma sample volume and without the need for prior EV purification. This proof-of-concept study demonstrates DEVA's potential for clinical utility to enhance prognosis as well as guide treatment for cancer.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

High-throughput single-cell, single-mitochondrial DNA assay using hydrogel droplet microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and intracellular heteroplasmy of mtDNA, but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95% mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Bentonite as a water-insoluble amorphous solid dispersion matrix for enhancing oral bioavailability of poorly water-soluble drugs.

Bentonite (BT), an orally administrable natural clay, is widely used for medical and pharmaceutical purposes due to its unique properties, including swelling, adsorption and ion-exchange. However, its application as a matrix of amorphous solid dispersion (ASD) formulations is rarely reported, despite the fact that drugs can adsorb to BT in an amorphous state. The objective of this study was to explore the feasibility of BT as a water-insoluble ASD matrix for enhancing the oral bioavailability of poorly water-soluble drugs, including sorafenib (SF). We prepared a novel BT-based ASD of an SF-BT composite (SFBTC) by adsorbing SF onto BT under acidic conditions using the ionic interaction between cationic SF and negatively charged BT. Scanning electron microscopy (SEM), powder X-ray diffractometry (pXRD), and differential scanning calorimetry (DSC) analyses revealed that SF adsorbed to BT in an amorphous state at SF:BT ratios from 1:3 to 1:10. In pharmacokinetic studies in rats, SFBTC (1:3) significantly improved the oral bioavailability of SF, and the AUClast of SFBTC (1:3) was 3.3-fold higher than that of NEXAVAR®, a commercial product of SF. An in vitro release study under sink conditions revealed that SFBTC (1:3) completely released SF in a pH-dependent manner, while a nonsink condition study indicated the generation of supersaturation under intestinal pH conditions. A kinetic solubility study showed that the release of SFBTC (1:3) followed the diffusion-controlled mechanism, which is a typical characteristic of water-insoluble matrix-based ASDs. The pharmacokinetic studies of drug-BT composites of various drugs belonging to BCS class II indicated that the pKa value of the adsorbed drugs is one of the most important factors determining their dissolution and oral bioavailability. These results suggest that BT could be a promising water-insoluble ASD matrix for improving the oral bioavailability of poorly water-soluble drugs, including SF.

Distribution and Phenotype of Goldenhar Syndrome and Its Association With Other Anomalies.

The purpose of this study was to investigate the distribution and phenotype of Goldenhar syndrome (GS) and its association with other anomalies. The samples consisted of 18 GS patients (6 males and 12 females; mean age at investigation, 7.4 ± 4.8 y) who were treated or followed up at the Department of Orthodontics, Seoul National University Dental Hospital between 1999 and 2021. The prevalence of side involvement and degree of mandibular deformity (MD), midface anomalies, and association with other anomalies were evaluated using statistical analysis. The prevalence of unilateral and bilateral MD did not differ (55.6% versus 44.4%). In unilateral MD cases, there was a tendency for higher prevalence of more severe Pruzansky-Kaban types than mild ones (type I, 10%; type IIa, 10%; type IIb, 50%; type III, 30%). Despite hypoplasia of the condyle/ramus complex, compensatory mandibular body growth occurred in 33.3% of GS patients (more severe side in bilateral MD cases, 37.5%, and ipsilateral side in unilateral MD cases, 30%). Class II molar relation was more prevalent than class I and class III molar relations (72.2% versus 11.1% versus 16.7%, P <0.01). Al total of 38.9% of patients had congenitally missing tooth. #7 facial cleft was found in 44.4% of patients. In midface anomalies, ear problem was the most common anomaly, followed by hypoplasia/absence of zygomatic arch and eye problem (88.9% versus 64.3% versus 61.1%, P <0.01). Association with the midface, spine, cardiovascular, and limb anomalies did not differ between unilateral and bilateral MD cases. These results might provide a basic guideline for diagnosis and treatment planning for GS patients.

Highly stable integration of graphene Hall sensors on a microfluidic platform for magnetic sensing in whole blood.

The detection and analysis of rare cells in complex media such as blood is increasingly important in biomedical research and clinical diagnostics. Micro-Hall detectors (µHD) for magnetic detection in blood have previously demonstrated ultrahigh sensitivity to rare cells. This sensitivity originates from the minimal magnetic background in blood, obviating cumbersome and detrimental sample preparation. However, the translation of this technology to clinical applications has been limited by inherently low throughput (<1 mL/h), susceptibility to clogging, and incompatibility with commercial CMOS foundry processing. To help overcome these challenges, we have developed CMOS-compatible graphene Hall sensors for integration with PDMS microfluidics for magnetic sensing in blood. We demonstrate that these graphene µHDs can match the performance of the best published µHDs, can be passivated for robust use with whole blood, and can be integrated with microfluidics and sensing electronics for in-flow detection of magnetic beads. We show a proof-of-concept validation of our system on a silicon substrate and detect magnetic agarose beads, as a model for cells, demonstrating promise for future integration in clinical applications with a custom CMOS chip.

Beads- and oil-free single molecule assay with immuno-rolling circle amplification for detection of SARS-CoV-2 from saliva.

Digital enzyme linked immunosorbent assays (ELISA) can be used to detect various antigens such as spike (S) or nucleocapsid (N) proteins of SARS-CoV-2, with much higher sensitivity compared to that achievable using conventional antigen tests. However, the use of microbeads and oil for compartmentalization in these assays limits their user-friendliness and causes loss of assay information due to the loss of beads during the process. To improve the sensitivity of antigen test, here, we developed an oil- and bead-free single molecule counting assay, with rolling circle amplification (RCA) on a substrate. With RCA, the signal is localized at the captured region of an antigen, and the signal from a single antigen molecule can be visualized using the same immune-reaction procedures as in the conventional ELISA. Substrate-based single molecule assay was theoretically evaluated for kd value, and the concentration of capture and detection antibodies. As a feasibility test, biotin-conjugated primer and mouse IgG conjugates were detected even at femto-molar concentrations with this digital immuno-RCA. Using this method, we detected the N protein of SARS-CoV-2 with a limit of detection less than 1 pg/mL more than 100-fold improvement compared to the detection using conventional ELISA. Furthermore, testing of saliva samples from COVID-19 patients and healthy controls (n = 50) indicated the applicability of the proposed method for detection of SARS-CoV-2 with 99.5% specificity and 90.9% sensitivity.

Implant-associated biofilm infection established in an experimental Galleria mellonella model.

The study reports in vivo biofilm infection implemented in an insect model. We mimicked implant-associated biofilm infections in Galleria mellonella larvae using toothbrush bristles and methicillin-resistant Staphylococcus aureus (MRSA). In vivo biofilm formation on bristle was achieved by sequentially injecting a bristle and MRSA into the larval hemocoel. It was found that biofilm formation was in progress without any external sign of infection in most of the bristle-bearing larvae for 12 h after MRSA inoculation. Whereas the activation of the prophenoloxidase system did not affect the preformed in vitro MRSA biofilms, an antimicrobial peptide interfered with in vivo biofilm formation when injected into bristle-bearing larvae infected with MRSA. Finally, our confocal laser scanning microscopic analysis revealed that the biomass of the in vivo biofilm is greater compared to that of the in vitro biofilm and harbors a distribution of dead cells, which might be bacteria and/or host cells.

On-chip microfluidic dual detection of amino acid metabolism disorders using cell-free protein synthesis.

Various metabolic diseases are associated with the accumulation of specific amino acids due to abnormal metabolic pathways, and thus can be diagnosed by measuring the level of amino acids in body fluids. However, present methods for amino acid analysis are not readily accessible because they require a complex experimental setup, expensive equipment, and a long processing time. Here, we present a dual sensing microfluidic device that enables fast, portable, and quantitative analysis of target amino acids, harnessing the biological mechanism of protein synthesis. In this device, the working principle of a finger-actuated pumping unit is applied, and the microchannels are designed to perform cell-free synthesis of a reporter protein in response to the target amino acids in the assay samples. Multiple steps required for the translational assay are controlled by the simple operation of two pushbuttons on the device. It is demonstrated that the developed microfluidic device provides precise quantification of two amino acids (methionine and phenylalanine) within 30 min at room temperature. We expect that the application of the presented device can be readily extended to the point-of-care testing of other metabolic compounds.

Effect of phosphatidylcholine in bentonite-quetiapine complex on enhancing drug release and oral bioavailability.

Bentonite (BT) is a biocompatible clay mineral that has advantageous properties as a pharmaceutical excipient. However, the application of BT in controlled-release oral formulations has been challenging due to incomplete drug release from BT-drug complexes. The objective of this study was to investigate the effect of modifying BT with zwitterionic phosphatidylcholine (PC) to enhance the dissolution of drugs, thereby increasing their oral bioavailability. Quetiapine (QTP) was chosen as a model drug, and the composition of the complex (BT-PC-QTP) was optimized to have the maximum QTP content and increase the total amount of QTP released. The in vitro release study showed that the incorporation of an appropriate amount of PC into BT improved the low release rate of the BT-QTP complex at pH 7.4, while the pH-dependent release property of BT was maintained. In an in vivo pharmacokinetic study in rats, the oral administration of the BT-PC-QTP complex showed significantly higher Cmax and AUC values than the BT-QTP complex. Moreover, BT-PC-QTP showed a 2.4-fold enhancement of oral bioavailability compared to the QTP powder group. The scanning electron microscopy (SEM), powder X-ray diffraction (pXRD), and differential scanning calorimetry (DSC) studies confirmed that the intercalation of PC and QTP into BT resulted in the adsorption of QTP in an amorphous state. The characterization of the nanoparticles generated from the BT-PC-QTP complex supported that PC enhanced the dissolution of QTP by forming nanosized PC particles. Taken together, the modification of BT with PC can be applied in pharmaceutical industry as a platform strategy to control the release of the BT-drug complex and enhance the oral bioavailability of poorly water-soluble drugs.

Bottom-up plasma-enhanced atomic layer deposition of SiO2 by utilizing growth inhibition using NH3 plasma pre-treatment for seamless gap-fill process.

The design-rule shrinkage in semiconductor devices is a challenge at every step of the integration process. In the gap-fill process for isolation, the seam and void formation cannot be suppressed by using a deposition process, which even has excellent step coverage. To achieve seamless gap fill in the high-aspect-ratio structure, which has a non-ideal etch profile such as a negative slope, the deposition process should be able to realize the "bottom-up growth" behavior. In this work, the bottom-up growth of a SiO2 plasma-enhanced atomic layer deposition (PE-ALD) process in a trench structure was investigated by using a growth inhibition process employing plasma treatment. N2 and NH3 plasma pre-treatments were employed to suppress the growth of the SiO2 PE-ALD process without any contamination, and the inhibition mechanism was investigated by performing surface chemistry analyses using X-ray photoelectron spectroscopy. Furthermore, the gap-fill characteristics of the SiO2 PE-ALD process were examined, depending on the process conditions of NH3 plasma pre-treatment, by performing cross-sectional field emission scanning electron microscopy measurements. Finally, a seamless gap-fill process in a high-aspect-ratio trench pattern was achieved by the bottom-up growth behavior of SiO2 PE-ALD using NH3 plasma pre-treatment.

Characterization of Treatment Modalities for Patients With Syndromic Craniosynostosis in Relation to Degree of Midface Hypoplasia and Patient's Age Using Longitudinal Follow-Up Data.

ABSTRACT: The purpose of this study was to investigate the type and frequency of use of treatment modalities (Tx-Mods) in patients with syndromic craniosynostosis (SC) using longitudinal follow-up data. A total of 28 patients with SC (24 Crouzon, 2 Apert, and 2 Antley-Bixler syndromes), who were treated at the Department of Orthodontics, Seoul National University Dental Hospital, Seoul, South Korea between 1998 and 2020, was included. According to the degree of midface hypoplasia (MH) at the initial visit (T1), the patients were divided into the mild-MH (78°≤SNA < 80°, n = 8), moderate-MH (76≤SNA < 78°, n = 7), and severe-MH (SNA < 76°, n = 13) groups. T1-age and Tx-Mods, including cal-varial surgery (CALS), orthopedic treatment (OPT), fixed orthodontic treatment, and midface advancement surgery in childhood (MAS-child) and adulthood (MAS-adult), were investigated. Complexity of MAS-adult was graded as follows: 0, no surgery; 1, orthognathic surgery; 2, distraction osteogenesis (DOG); 3, combination of distraction osteogenesis and orthognathic surgery. Then, statistical analysis was performed. Percentage distribution of Tx-Mods was 71.4% in CALS, 21.4% in MAS-child, 42.9% in OPT, 100% in fixed orthodontic treatment, and 89.3% in MAS-adult. 92.9% of patients underwent MAS more than once. The number of MAS increased according to the severity of MH ( P < 0.05). The complexity of MAS-adult increased as T1-age and severity of MH increased (all P < 0.05); whereas it decreased when CALS and OPT were performed (all P < 0.05). However, MAS in childhood did not guarantee the avoidance of additional MAS in adulthood ( P > 0.05). These findings may be used as basic guidelines for successful treatment planning and prognosis prediction in patients with SC.

Subcutaneously Injectable Hyaluronic Acid Hydrogel for Sustained Release of Donepezil with Reduced Initial Burst Release: Effect of Hybridization of Microstructured Lipid Carriers and Albumin.

The daily oral administration of acetylcholinesterase (AChE) inhibitors for Alzheimer's disease features low patient compliance and can lead to low efficacy or high toxicity owing to irregular intake. Herein, we developed a subcutaneously injectable hyaluronic acid hydrogel (MLC/HSA hydrogel) hybridized with microstructured lipid carriers (MLCs) and human serum albumin (HSA) for the sustained release of donepezil (DNP) with reduced initial burst release. The lipid carrier was designed to have a microsized mean diameter (32.6 ± 12.8 µm) to be well-localized in the hydrogel. The hybridization of MLCs and HSA enhanced the structural integrity of the HA hydrogel, as demonstrated by the measurements of storage modulus (G'), loss modulus (G″), and viscosity. In the pharmacokinetic study, subcutaneous administration of MLC/HSA hydrogel in rats prolonged the release of DNP for up to seven days and reduced the initial plasma concentration, where the Cmax value was 0.3-fold lower than that of the control hydrogel without a significant change in the AUClast value. Histological analyses of the hydrogels supported their biocompatibility for subcutaneous injection. These results suggest that a new hybrid MLC/HSA hydrogel could be promising as a subcutaneously injectable controlled drug delivery system for the treatment of Alzheimer's disease.

Pushbutton-activated microfluidic cartridge as a user-friendly sample preparation tool for diagnostics.

Microfluidic technologies have several advantages in sample preparation for diagnostics but suffer from the need for an external operation system that hampers user-friendliness. To overcome this limitation in microfluidic technologies, a number of user-friendly methods utilizing capillary force, degassed poly(dimethylsiloxane), pushbutton-driven pressure, a syringe, or a pipette have been reported. Among these methods, the pushbutton-driven, pressure-based method has a great potential to be widely used as a user-friendly sample preparation tool for point-of-care testing or portable diagnostics. In this Perspective, we focus on the pushbutton-activated microfluidic technologies toward a user-friendly sample preparation tool. The working principle and recent advances in pushbutton-activated microfluidic technologies are briefly reviewed, and future perspectives for wide application are discussed in terms of integration with the signal analysis system, user-dependent variation, and universal and facile use.

Pushbutton-activated microfluidic dropenser for droplet digital PCR.

Here, we report a portable microfluidic device to generate and dispense droplets simply operated by pushbutton for droplet digital polymerase chain reaction (ddPCR), which is named pushbutton-activated microfluidic dropenser (droplet dispenser) (PAMD). After loading the PCR mixtures and the droplet generation oil to PAMD, digitized PCR mixtures are prepared in PCR tubes after the actuation of a pushbutton. Multiple droplet generation units are simultaneously operated by a single pushbutton, and the size of droplets is controllable by adjusting the geometry of the droplet generation channel. To examine the performance of PAMD, digitized PCR mixtures containing genomic DNA of Escherichia coli (E. coli) O157:H7 prepared by PAMD were assessed by a fluorescence signal analyzer after PCR with a thermal cycler. As a result, PAMD can produce analytical droplets for ddPCR as much as a conventional droplet generator even though any external equipment is not required.

Hyaluronidase Inhibitor-Incorporated Cross-Linked Hyaluronic Acid Hydrogels for Subcutaneous Injection.

Hyaluronidase (HAase) inhibitor-incorporated hyaluronic acid (HA) hydrogel cross-linked with 1,4-butanediol diglycidyl ether (BDDE) was designed to reduce the toxicity risk induced by BDDE and its biodegradation rate in subcutaneous tissue. The formulation composition of hydrogel and its preparation method were optimized to have a high swelling ratio and drug content. Quercetin (QCT) and quetiapine (QTP), as an HAase inhibitor and model drug, respectively, were incorporated into the cross-linked hydrogel using the antisolvent precipitation method for extending their release after subcutaneous injection. The cross-linked HA (cHA)-based hydrogels displayed appropriate viscoelasticity and injectability for subcutaneous injection. The incorporation of QCT (as an HAase inhibitor) in the cHA hydrogel formulation resulted in slower in vitro and in vivo degradation profiles compared to the hydrogel without QCT. Single dosing of optimized hydrogel injected via a subcutaneous route in rats did not induce any acute toxicities in the blood chemistry and histological staining studies. In the pharmacokinetic study of rats following subcutaneous injection, the cHA hydrogel with QCT exhibited a lower maximum QTP concentration and longer half-life and mean residence time values compared to the hydrogel without QCT. All of these results support the designed HAase inhibitor-incorporated cHA hydrogel being a biocompatible subcutaneous injection formulation for sustained drug delivery.

Preparation and characterization of sorafenib-loaded microprecipitated bulk powder for enhancing oral bioavailability.

The aim of this study was to prepare and evaluate Eudragit-based microprecipitated bulk powder (MBP) formulations to enhance the oral bioavailability of sorafenib. Cationic Eudragit E PO and anionic Eudragit S100 were selected for MBP preparation. Ursodeoxycholic acid (UDCA)-incorporated MBP was also prepared to study the synergistic effect of UDCA in enhancing the bioavailability of sorafenib. Sorafenib-loaded MBPs were successfully prepared by a pH-controlled precipitation method using an aqueous antisolvent. Submicron-sized particles of MBPs were observed by scanning electron microscopy, and the amorphous form of sorafenib in MBPs was confirmed by powder X-ray diffraction. MBPs of cationic and anionic Eudragits showed different in vitro dissolution and pharmacokinetic profiles in rats. Sorafenib in Eudragit E PO-based MBP (E PO-MBP) was rapidly dissolved at low pH conditions (pH 1.2 and 4.0), but was precipitated again at pH 4.0 within 4 h. Dissolution of sorafenib from Eudragit S100-based MBP (S100-MBP) was high at pH 7.4 and did not precipitate for up to 4 h. After oral administration to rats, all MBPs, compared with powder, improved the oral absorption of sorafenib, with S100-MBP showing 1.5-fold higher relative oral bioavailability than E PO-MBP. Moreover, incorporation of UDCA in S100-MBP (S100-UDCA-MBP) further increased the Cmax and oral bioavailability of sorafenib, although the dissolution was not significantly different from that of S100-MBP. Taken together, Eudragit-based MBP formulations could be a promising strategy for enhancing the oral bioavailability of sorafenib.

Microfluidic channel-integrated hanging drop array chip operated by pushbuttons for spheroid culture and analysis.

Although the hanging drop methods have a number of advantages for spheroid culture, they suffer from reagent exchange procedures that depend on tedious and accurate liquid handling by manual pipetting or robotic arms. To simplify these procedures, we developed a method for liquid handling in a hanging drop array (HDA) chip for spheroid culture and analysis by integrating microfluidic channels operated by pushbuttons. Six finger-actuated microfluidic pumping units connected to a 3 × 3 HDA can draw or replenish reagents in an HDA chip without any external equipment. The initial cell seeding, medium exchange, and staining for further analysis can be simply done by pushing the buttons in the programmed order. After the assessment of the reagent exchange ratio of the device, BT474 spheroids of various sizes were cultured in the device for 7 days by exchanging the medium once a day and stained on the same device by exchanging the medium with staining reagents for the LIVE/DEAD assay. Furthermore, the cultured spheroids were embedded into collagen by exchanging the medium with a collagen solution to mimic a cancer metastasis environment.

Reciprocating flow-assisted nucleic acid purification using a finger-actuated microfluidic device.

Molecular diagnostics can provide a powerful diagnostic tool since it can detect pathogens with high sensitivity, but complicated sample preparation procedures limit its widespread use as an on-site detection tool that relies on the skilled person and external equipment. To resolve these limitations, we report a solid-phase nucleic acid purification using a finger-actuated microfluidic device, which can control a set amount of flow regardless of differences in end-users. To increase the recovery rate, a finger-actuated reciprocator was newly developed and integrated into the microfluidic device that can efficiently react with silica microbeads and reagents. After verifying the finger-actuated microfluidic reciprocator, the effect of the reciprocating flow on the recovery rate was assessed to purify the standard DNA of the hepatitis B virus (HBV). The recovery rate was increased up to ∼50% and 955 to 955 000 IU mL-1 of HBV standard DNA was successfully purified and detected by a real-time polymerase chain reaction. Furthermore, the proposed microfluidic device was exploited to purify the HBV DNA from the patient's blood plasma samples.

Colorimetric Detection of Escherichia coli O157:H7 with Signal Enhancement Using Size-Based Filtration on a Finger-Powered Microfluidic Device.

Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound Escherichia coli (E. coli) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound E. coli O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 102 CFU/mL E. coli O157:H7 could be detected by the enhanced colorimetric signal on a proposed device.