RESUMO
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 â¼50 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.
Assuntos
Aptâmeros de Nucleotídeos , Infecções Relacionadas à Prótese , Técnica de Seleção de Aptâmeros , Líquido Sinovial , alfa-Defensinas , alfa-Defensinas/análise , Humanos , Infecções Relacionadas à Prótese/diagnóstico , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , Líquido Sinovial/química , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodosRESUMO
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.
Assuntos
Vesículas Extracelulares , Microfluídica , Vesículas Extracelulares/metabolismo , RNARESUMO
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.
Assuntos
Técnicas Biossensoriais , Doenças Cardiovasculares , Humanos , Doenças Cardiovasculares/diagnóstico , Microfluídica , Biomarcadores , Peptídeo Natriurético Encefálico , Proteína C-Reativa , Fibrinogênio , Fragmentos de PeptídeosRESUMO
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.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Biomarcadores Tumorais/análise , Bile/química , Bile/metabolismo , Microfluídica , Método Simples-Cego , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/diagnóstico , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologiaRESUMO
Variants of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) have evolved such that it may be challenging for diagnosis and clinical treatment of the pandemic coronavirus disease-19 (COVID-19). Compared with developed SARS-CoV-2 diagnostic tools recently, aptamers may exhibit some advantages, including high specificity/affinity, longer shelf life (vs. antibodies), and could be easily prepared. Herein an integrated microfluidic system was developed to automatically carry out one novel screening process based on the systematic evolution of ligands by exponential enrichment (SELEX) for screening aptamers specific with SARS-CoV-2. The new screening process started with five rounds of positive selection (with the S1 protein of SARS-CoV-2). In addition, including non-target viruses (influenza A and B), human respiratory tract-related cancer cells (adenocarcinoma human alveolar basal epithelial cells and dysplastic oral keratinocytes), and upper respiratory tract-related infectious bacteria (including methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae), and human saliva were involved to increase the specificity of the screened aptamer during the negative selection. Totally, all 10 rounds could be completed within 20 h. The dissociation constant of the selected aptamer was determined to be 63.0 nM with S1 protein. Limits of detection for Wuhan and Omicron clinical strains were found to be satisfactory for clinical applications (i.e. 4.80 × 101 and 1.95 × 102 copies/mL, respectively). Moreover, the developed aptamer was verified to be capable of capturing inactivated SARS-CoV-2 viruses, eight SARS-CoV-2 pseudo-viruses, and clinical isolates of SARS-CoV-2 viruses. For high-variable emerging viruses, this developed integrated microfluidic system can be used to rapidly select highly-specific aptamers based on the novel SELEX methods to deal with infectious diseases in the future.
Assuntos
Aptâmeros de Nucleotídeos , COVID-19 , Staphylococcus aureus Resistente à Meticilina , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Microfluídica , Teste para COVID-19 , Staphylococcus aureus Resistente à Meticilina/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/metabolismo , Técnica de Seleção de Aptâmeros/métodosRESUMO
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.
Assuntos
Artrite Infecciosa , Infecções Relacionadas à Prótese , Humanos , Líquido Sinovial/química , Infecções Relacionadas à Prótese/diagnóstico , Microfluídica , Sensibilidade e Especificidade , Biomarcadores/metabolismo , Proteína C-Reativa/análise , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/metabolismoRESUMO
The diagnosis of periprosthetic joint infection (PJI) remains a labor-intensive and challenging issue, with life-threatening complications associated with misdiagnoses. Superior diagnostic approaches are therefore urgently needed, and synovial biomarkers are gaining substantial attention in this capacity. A new aptamer-based sandwich assay was developed where the aptamer probes specific to one such biomarker, alpha-defensin human neutrophil protein 1 (HNP 1), was integrated herein into a new microfluidic platform. The magnetic beads coated with the primary aptamer probe were able to bind the target protein with high affinity and high specificity in synovial fluid and a fluorescent-labelled secondary aptamer were further used to quantify HNP 1 in a sandwich approach. Up to four clinical samples with low volume (â¼50 µL each) in a much faster assay including detection within <60 min with 100% accuracy (with totally 13 clinical samples without the need of sample pretreatment) through the use of the aptamer-based sandwich assay were automatically detected on a single chip. The wide dynamic range of this compact device, 0.5-100 mg/L, highlights its utility for future PJI diagnostics in the clinic.
Assuntos
Artrite Infecciosa , Técnicas Biossensoriais , alfa-Defensinas , Humanos , Microfluídica , Neutrófilos , Biomarcadores , Oligonucleotídeos , Líquido Sinovial , Artrite Infecciosa/diagnóstico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Total joint arthroplasty (TJA) has significantly improved the quality of life for millions suffering from end-stage arthritis. However, periprosthetic joint infections (PJI) remain a serious complication, necessitating extensive interventions and prolonged antimicrobial treatments. The aging population is expected to lead to a rise in TJA cases, subsequently increasing the incidence of PJI, particularly in the elderly who face higher mortality rates. Current diagnostic methods for suspected PJI, such as radiographs and biochemical markers like CRP and ESR, exhibit limited sensitivity. Therefore, there is a critical need for a specific synovial fluid biomarker assay to enhance PJI diagnosis using specific SF-based assay. RESULTS: This study introduces a novel microfluidic chip with a paper-based aptamer-sandwich assay for the quantitative detection of HNP 1, a crucial PJI biomarker, in synovial fluid. The assay leverages the advantages of aptamers over antibodies, demonstrating high selectivity and affinity for target molecules. The integration of a nitrocellulose (NC) membrane onto the microfluidic platform represents a significant advancement, reducing background signals and simplifying the assay procedure without intricate procedure and pre-treatment. The NC membrane-based microfluidic device offers rapid, cost-effective, and highly sensitive detection of HNP 1, with a limit of detection of 0.5 mg L-1. The microfluidic device demonstrates exceptional performance, detecting up to four clinical samples in approximately 42 min on a single chip with 100 % accuracy, as confirmed by analysis of 12 clinical samples and comparison with "gold-standard". Moreover, the assay exhibits a wide dynamic range of 0.5-100 mg L-1, underscoring its potential as a powerful tool for PJI diagnosis in clinical settings. SIGNIFICANCE: This work introduces a paper-based microfluidic system tailored for rapid HNP 1 detection using synovial fluid near joint region (and not serum via blood) for better diagnosis. The innovative paper-based aptamer-sandwich assay yields results within 42-min. Significantly, it boasts a wide dynamic range, detecting levels from an impressive 0.5 mg L-1, crucial in the 2.6 mg L-1 threshold region. This heightened sensitivity and expansive detection capability establish our assay as a leader in PJI diagnostics, promising unmatched precision and efficiency in clinical applications.
Assuntos
Aptâmeros de Nucleotídeos , Biomarcadores , Dispositivos Lab-On-A-Chip , Papel , Infecções Relacionadas à Prótese , Líquido Sinovial , alfa-Defensinas , Humanos , Aptâmeros de Nucleotídeos/química , Biomarcadores/análise , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Infecções Relacionadas à Prótese/diagnóstico , Líquido Sinovial/química , alfa-Defensinas/análiseRESUMO
Rapid, sensitive and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of great need for effective quarantining and treatment. Real-time reverse-transcription polymerase chain reaction requiring thermocyling has been commonly used for diagnosis of SARS-CoV-2 though it may take two to 4 h before lengthy sample pretreatment process and require bulky apparatus and well-trained personnel. Since multiple reverse transcription loop-mediated isothermal amplification (multiple RT-LAMP) process without thermocycling is sensitive, specific and fast, an electromagnetically-driven microfluidic chip (EMC) was developed herein to lyse SARS-CoV-2 viruses, extract their RNAs, and perform qualitative analysis of three marker genes by on-chip multiple RT-LAMP in an automatic format within 82 min at a limit of detection of only â¼5000 copies per reaction (i.e. 200 virus/ µL). This compact EMC may be especially promising for SARS-CoV-2 diagnostics in resource-limited countries.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Técnicas de Laboratório Clínico , Humanos , Microfluídica , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , RNA Viral/genética , Transcrição Reversa , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Ácidos Nucleicos Livres , Microfluídica , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/genética , Feminino , Humanos , Microfluídica/métodos , Mutação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
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.
Assuntos
Biomarcadores Tumorais , Ácidos Nucleicos Livres , Microfluídica , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/isolamento & purificação , Metilação de DNA , Análise de Sequência com Séries de Oligonucleotídeos , PrognósticoRESUMO
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.
RESUMO
Periprosthetic joint infections (PJIs) arising from joint arthroplasty are dreadful, yet difficult to diagnose in subtle cases. Definite diagnosis requires microbiological culture to confirm the causative pathogens. However, up to 40% of culture-negative PJI needs other surrogate biomarkers such as human neutrophil peptide 1 (HNP 1) to improve diagnostic accuracy or gauge therapeutic responses. To devise a diagnostic method, systematic evolution of ligands by exponential enrichment (SELEX) (five rounds) was used to screen PJI biomarkers on a compact (20 × 20 × 35 cm), integrated microfluidic system equipped with two separate Peltier devices and one magnetic control module where an aptamer with high affinity and specificity for HNP 1, which has been used as one of the synovial fluid (SF) biomarkers for detecting PJI, was identified for the first time. Two rounds of negative selection (with immunoglobulin G & human serum album) on-chip followed by one round of unique "competitive selection" with SF extracted from PJI patients validated the specificity of the HNP 1 aptamer. The dissociation constant was measured to be 19 nM. The applicability of SF HNP 1 levels for diagnosing PJI was then verified by a new aptamer-based enzyme-linked immunosorbent assay (ELISA)-like assay. It is envisioned that this new aptamer and the associated assay could be used in future clinical applications.
Assuntos
Infecções Relacionadas à Prótese , alfa-Defensinas , Biomarcadores/análise , Humanos , Microfluídica , Infecções Relacionadas à Prótese/diagnóstico , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/cirurgia , Sensibilidade e Especificidade , Líquido Sinovial/química , alfa-Defensinas/análiseRESUMO
Ovarian cancer (OvCa) is the most fatal among gynecological cancers and affects many women worldwide. Since OvCa is prone to metastasis, which significantly increases chances of death, biomarkers for early-stage OvCa are greatly needed. This study develops an integrated microfluidic platform for isolating and quantifying one of the OvCa blood biomarkers. As a demonstration, microRNA-21 (miRNA-21), which is one of the important biomarkers for cancers, was isolated and measured in this study. Extracellular vesicles (EVs) in blood were first captured and isolated by anti-CD63-coated magnetic beads. Then, EV-encapsulated miRNA-21 was isolated by complementary DNA-coated magnetic beads, and finally the isolated miRNA-21 was quantified by digital polymerase chain reaction (digital PCR, dPCR). The integrated chip featured a sample treatment module and a miRNA quantification module that automated the entire process, and the limit of detection (LOD) was 11 copies per mL. The inaccuracy of the miRNA quantification module (i.e. dPCR) was found to be <12%. Additionally, spiked samples and clinical samples were used to test the performance of the developed platform. It is envisioned that the developed system can serve as a valuable and promising tool for OvCa biomarker measurements.
Assuntos
Vesículas Extracelulares , MicroRNAs , Biomarcadores Tumorais , Humanos , Separação Imunomagnética , MicroRNAs/genética , MicrofluídicaRESUMO
In this work we developed an integrated microfluidic system for automatically detecting the ovarian clear cell carcinoma (OCCC) biomarker FXYD2. Dealing with ascites from ovarian cancer patients, capture of cancer cells, isolation of messenger RNA, and quantitative reverse-transcription polymerase chain reaction were integrated into a single microfluidic chip and carried out on-chip automatically. OCCC is a subtype of ovarian cancer with a high mortality risk, and a high FXYD2 gene expression level was shown to be closely associated with OCCC. The lowest limit of quantification using a benchtop protocol of this system could be as low as 100 copies per sample. By normalizing the expression to a housekeeping gene, GAPDH, a simple cycle threshold ratio index could distinguish high FXYD2 expression cells from the low-expression ones. This developed platform may therefore facilitate future OCCC diagnosis and/or prognosis.
Assuntos
Adenocarcinoma de Células Claras , Neoplasias Ovarianas , Feminino , Humanos , Microfluídica , Prognóstico , RNA Mensageiro , ATPase Trocadora de Sódio-PotássioRESUMO
Antimicrobial resistance stemming from indiscriminate usage of antibiotics has emerged as a global healthcare issue with substantial economic implications. The inefficacy of commonly used antibiotics combined with superfluous consumption has worsened the issue. Rapid antimicrobial susceptibility testing (AST) to antibiotics can be advantageous in thwarting bacterial infections. Therefore, this study developed a simple nanoliter array-based microfluidic platform for performing rapid AST, which can handle and manipulate liquids both in nanoliter and microliter volumes. The platform consisted of two microfluidic devices, one for performing AST and another for diluting antibiotics and these two were suitably integrated. The microfluidic device used for generating microarrays for AST experiments is single-layered (no air layer) and has no active microvalves and air hole, which makes the device easy to fabricate and use. The loading process ensures uniform distribution of bacteria and relies on displacing the air from microarrays through porous polydimethylsiloxane membranes. Furthermore, the chip for dilution consisted of active microfluidic components, and could prepare and test seven different concentrations of antibiotics, which make the platform multiplexed and be capable of evaluating minimum inhibitory concentrations (MICs), a clinically relevant parameter. MIC determination requires less number of bacteria (â¼2000) and hence shortens the pre-culture step, i.e. bacteria culture in blood and urine. This automated system demonstrated AST and evaluated MICs using Escherichia coli and two antibiotics, including ampicillin and streptomycin, and the results were ascertained using a gold standard method. It only took 8-9 h to perform AST, which is substantially less compared to a conventional process and hence is of high clinical utility.
Assuntos
Antibacterianos , Microfluídica , Antibacterianos/farmacologia , Escherichia coli , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade MicrobianaRESUMO
Cell-released, membrane-encapsulated extracellular vesicles (EVs) serve as a means of intercellular communication by delivering bioactive cargos including proteins, nucleic acids and lipids. EVs have been widely used for a variety of biomedical applications such as biomarkers for disease diagnosis and drug delivery vehicles for therapy. Herein, this study reports a novel method for label-free, contact-free isolation and recovery of EVs via optically-induced dielectrophoresis (ODEP) on a pneumatically-driven microfluidic platform with minimal human intervention. At an optimal driving frequency of 20 kHz and a voltage of 20 Vpp, an ODEP force from a 75 µm moving light beam was characterized to be 23.5-97.7 fN in 0.2 M sucrose solution. Furthermore, rapid enrichment of EVs with a small volume of only 27 pL in 32 s achieved an increase of 272-fold by dynamically shrinking circular light patterns. Moreover, EVs could be automatically isolated and recovered within 25 min, while achieving a releasing efficiency of 99.8% and a recovery rate of 52.2% by using an integrated microfluidics-based optically-induced EV isolation (OIEV) platform. Given the capacity of label-free, contact-free EV isolation, and automatic, easy-releasing EV recovery, this integrated OIEV platform provides a unique approach for EV-based disease diagnosis and drug delivery applications.
Assuntos
Vesículas Extracelulares , Ácidos Nucleicos , Comunicação Celular , Humanos , Lipídeos , MicrofluídicaRESUMO
Ovarian cancer (OvCa) is the most lethal gynecologic malignancy owing to its high chemoresistance and late diagnosis, which lead to a poor prognosis. Hence, developing new therapeutic modalities is important for OvCa patient treatment. Our previous results indicated that a novel aptamer, Tx-01, can specifically recognize serous carcinoma cells and tissues. Here, we aim to clarify the clinical role and possible molecular mechanisms of Tx-01 in OvCa. Immunostaining and statistical analysis were performed to detect the interaction of Tx-01 and heat shock protein 70/Notch1 intracellular domain (HSP70/NICD) in OvCa. The in vitro and in vivo experiments were carried out to demonstrate the potential mechanisms of Tx-01. Results show that Tx-01 reduced serous OvCa OVCAR3 cell migration and invasion and inhibited HSP70 nuclear translocation by interrupting the intracellular HSP70/NICD interaction. Furthermore, Tx-01 suppressed serous-type OVCAR3 cell tumor growth in vivo. Tx-01 acts as a prognostic factor through its interaction with membrane-bound HSP70 (mHSP70 that locates on the cell surface without direct interaction to NICD) on ascitic circulating tumor cells (CTCs) and is reported to be involved in natural killer (NK) cell recognition and activation. Our data demonstrated that Tx-01 interacted with HSP70 and showed therapeutic and prognostic effects in serous OvCa. Tx-01 might be a potential inhibitor for use in serous OvCa treatment.
RESUMO
Urinary tract infections (UTIs) are bacterial infections that 1) commonly affect females, 2) can pose high risks to impair kidney function, 3) are often treated with broad-spectrum antibiotics, and 4) are associated with high recurrence rates due to the evolution of drug-resistant strains. To choose the appropriate antibiotic, the minimum inhibitory concentration (MIC) among a panel of antibiotics should be determined before administration to avoid inadequate dosing or use of wrong antibiotics. To meet with the unmet needs, we developed a bead-based method for bacterial preconcentration with capture rates ranging from 20-50% and then automatically performed on-chip AST on an automated device which was composed of a pneumatic control module, a temperature control module and a chip image processing module. The developed portable system was capable of automatically conducting AST and MIC measurements using urine samples (via image analysis) in only 4.5-9 h and tested on four common UTIs bacterial strains. This compact system may therefore be promising for point-of-care personalized medicine in the near future.
Assuntos
Infecções Urinárias , Antibacterianos/farmacologia , Bactérias , Farmacorresistência Bacteriana , Feminino , Humanos , Testes de Sensibilidade Microbiana , Infecções Urinárias/diagnóstico , Infecções Urinárias/tratamento farmacológicoRESUMO
Cancer is among the world's most deadly inflictions, and early diagnosis is critical. Aptamers have shown utility as cancer probes since they can be screened rapidly in vitro against cancer tissues using systematic evolution of ligands by exponential enrichment (SELEX) process. However, bench-top SELEX procedures are relatively labor-intensive and time-consuming; ideally, they could instead be carried out on microfluidic devices, yet this requires optimization of buffer and reaction conditions. Herein an integrated microfluidic system (IMS) was established to automatically carry out the optimization of aptamer selection. A "formulation chip" was developed that could mix salt solutions at differing final concentrations, and the resulting optimal binding buffer was transferred to another "optimization-SELEX chip" for the following tissue-SELEX. Two aptamers were successfully screened; one of which, H-45, exhibited high specificity and affinity towards ovarian cancer tissue samples, suggesting that this IMS might be a promising device for screening of cancer associated aptamers for cancer diagnosis.