RESUMEN
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.
Asunto(s)
Infecciones Relacionadas con Prótesis , alfa-Defensinas , Biomarcadores/análisis , Humanos , Microfluídica , Infecciones Relacionadas con Prótesis/diagnóstico , Infecciones Relacionadas con Prótesis/microbiología , Infecciones Relacionadas con Prótesis/cirugía , Sensibilidad y Especificidad , Líquido Sinovial/química , alfa-Defensinas/análisisRESUMEN
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.
Asunto(s)
Antibacterianos , Microfluídica , Antibacterianos/farmacología , Escherichia coli , Dispositivos Laboratorio en un Chip , Pruebas de Sensibilidad MicrobianaRESUMEN
The search of alternative approaches to epithelial cell adhesion molecule (EpCAM), for the isolation of circulating tumor cells (CTC), is on the rise. This work attempts at evaluating the feasibility of using a new glycosaminoglycan, SCH45, as a probe to isolate CTCs from the peripheral blood of 65 advanced/metastatic cholangiocarcinoma (CCA) patients. The positive enrichment of CTCs from 1 mL of blood using SCH45-bound magnetic beads and subsequent staining on an integrated microfluidic platform is demonstrated. Results detailing CTC concentrations averaging ≥1 CTCs mL-1 of blood are shown, and a conventional protein biomarker, EpCAM, has been used to corroborate the finding that 100% of the patients possess CTCs in their blood. Studies detailing the use of CTCs in the prognostic monitoring and treatment effectiveness of advanced/metastatic CCA are scarce, and the isolation of CTCs from all CCA patients tested has not been reported yet. A strong correlation between CTC counts and disease progression at the time of and/or in advance of radiographic imaging in patients receiving chemotherapy is also reported. This study is one of its kind with the new probe and reduced sample volume and has potential for use in CCA diagnosis and prognosis in the near future.
Asunto(s)
Neoplasias de los Conductos Biliares , Colangiocarcinoma , Células Neoplásicas Circulantes , Conductos Biliares Intrahepáticos , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial , Glicosaminoglicanos , Humanos , MicrofluídicaRESUMEN
In this study, an enzyme linked DNA aptamer based assay was optimized for human cardiac troponin I (cTnI) detection which is a prominent biomarker for acute myocardial infarction (AMI), on an integrated microfluidic platform. This platform allowed for the multiplex detection of six samples (5 µL per sample), and only 30 min were required for detection. First, cTnI-specific aptamers were surface-coated on magnetic beads. Bead-captured proteins were allowed to bind to a primary cTnI antibody and then to a secondary antibody labelled with horseradish peroxidase. Finally, chemiluminescence intensities were detected for quantification of cTnI. Purified proteins, serum from AMI patients and unknown serum samples were used to test the efficacy of the on-chip system. The limit of detection was measured to be only 12 ng L-1, and off-target effects from other proteins were minimal. This sensitive, cTnI-specific aptamer-based assay could consequently be used for reliable diagnosis of AMI.
Asunto(s)
Técnicas Biosensibles/métodos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Troponina I/sangre , Aptámeros de Nucleótidos/química , Armoracia/enzimología , Secuencia de Bases , Biomarcadores/sangre , ADN/química , Enzimas Inmovilizadas/química , Peroxidasa de Rábano Silvestre/química , Humanos , Separación Inmunomagnética/métodos , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , Reproducibilidad de los ResultadosRESUMEN
Although cardiovascular diseases such as heart failure (HF) affect 30 million people globally, the early detection of HF has, until recently, been difficult and prone to misdiagnoses. Monitoring the circulatory levels of a relatively new biomarker, the N-terminal prohormone of a B-type natriuretic peptide, could be used for early risk evaluation of HF. Therefore, we developed a pneumatically-driven, automatic integrated microfluidic platform equipped with micromixers, micropumps, and microvalves for the simultaneous detection of NT-proBNP in up to six clinical samples within 25 min by using a novel aptamer-based sandwich assay, and the limit of detection was only 1.53 pg mL-1; given that the chip is 64% more compact than those developed in our prior works and requires only 5 µL of sample input, it may serve as a promising tool for early diagnosis of HF.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/instrumentación , Dispositivos Laboratorio en un Chip , Péptido Natriurético Encefálico/análisis , Fragmentos de Péptidos/análisis , Integración de Sistemas , Aptámeros de Nucleótidos/genética , Secuencia de Bases , Calibración , Diseño de Equipo , Humanos , Límite de Detección , Péptido Natriurético Encefálico/metabolismo , Fragmentos de Péptidos/metabolismo , Factores de TiempoRESUMEN
Certain blood-borne biomarkers offer a potent methodology for understanding the risk of cardiovascular diseases (CVDs) with clinicians generally advocating the use of multiple biomarkers for proper risk assessment of CVDs. Herein four such CVDs biomarkers- C-reactive protein (CRP), N-terminal pro b-type natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), and fibrinogen- were rapidly (5â¯min) analyzed from clinical samples (~â¯4⯵L) on an integrated microfluidic platform equipped with 1) immobilized highly specific aptamer probes and 2) field-effect transistor (FET)-based sensor arrays. The calibration curve from the FET sensor arrays showed good agreement in the physiological concentration ranges for CRP (0.1-50â¯mg/L), NT-proBNP (50-10,000â¯pg/mL), cTnI (1-10,000â¯pg/mL), and fibrinogen (0.1-5â¯mg/mL). The developed prototype of this fully automated portable device requires minimal reagent and sample inputs and consequently shows great promise for next-generation point-of-care devices assaying multiple CVDs biomarkers in clinical samples.
Asunto(s)
Técnicas Biosensibles/instrumentación , Proteína C-Reactiva/análisis , Enfermedades Cardiovasculares/sangre , Fibrinógeno/análisis , Dispositivos Laboratorio en un Chip , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Troponina I/sangre , Aptámeros de Nucleótidos/química , Biomarcadores/sangre , Diseño de Equipo , Humanos , Límite de Detección , Sistemas de Atención de Punto , Transistores ElectrónicosRESUMEN
As cardiovascular diseases (CVD) are responsible for millions of deaths annually, there is a need for rapid and sensitive diagnosis of CVD at earlier stages. Aptamers generated by systematic evolution of ligands by exponential enrichment (SELEX) processes have been shown to be superior to conventional antibody-based cardiac biomarker detection. However, SELEX is a complicated, lengthy procedure requiring multiple rounds of extraction/amplification and well-trained personnel. To circumvent such issue, we designed an automated, miniaturized SELEX platform for the screening of aptamers towards three protein biomarkers associated with CVDs: N-terminal pro-peptide of B-type natriuretic peptide, human cardiac troponin I, and fibrinogen. The developed microfluidic platform was equipped with microfluidic devices capable of sample transport and mixing along with an on-chip nucleic acid amplification module such that the entire screening process (5 rounds of selection in 8â¯h.) could be performed consecutively on a single chip while consuming only 35⯵L of reagents in each cycle. This system may therefore serve as a promising, sensitive, cost-effective platform for the selection of aptamers specific for CVD biomarkers.
Asunto(s)
Aptámeros de Nucleótidos/química , Enfermedades Cardiovasculares/diagnóstico , Dispositivos Laboratorio en un Chip , Técnica SELEX de Producción de Aptámeros/instrumentación , Biomarcadores/análisis , Técnicas Biosensibles/instrumentación , Diseño de Equipo , Fibrinógeno/análisis , Humanos , Péptido Natriurético Encefálico/análisis , Fragmentos de Péptidos/análisis , Troponina I/análisisRESUMEN
An integrated microfluidic system capable of automatically identifying aptamers specific to cholangiocarcinoma (CCA) cells was developed herein. The developed system was capable of performing cell-based systematic evolution of ligands via an exponential enrichment (Cell-SELEX) process on-chip, and only six rounds of Cell-SELEX were required to identify high specificity aptamers; this represents a significant improvement in speed over conventional SELEX, in which 15-20 rounds are typically required. Using the microfluidic chip developed, three aptamers specific to CCA cells (one for SNU-478 cells and two for HuCCT-1 cells) were successfully screened. This automated system could be modified to uncover aptamer probes against other cancer cells, thereby allowing for earlier diagnosis and consequently a potentially improved prognosis.
RESUMEN
Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.