RESUMEN
Strategies for rapid, effective nucleic acid processing hold tremendous significance to the clinical analysis of circulating tumor DNA (ctDNA), a family of important markers indicating tumorigenesis and metastasis. However, traditional techniques remain challenging to achieve efficient DNA enrichment, further bringing about complicated operation and limited detection sensitivity. Here, we developed an ion concentration polarization microplatform that enabled highly rapid, efficient enrichment and purification of ctDNA from a variety of clinical samples, including serum, urine, and feces. The platform demonstrated efficiently separating and enriching ctDNA within 30 s, with a 100-fold improvement over traditional methods. Integrating an on-chip isothermal amplification module, the platform further achieved 100-fold enhanced sensitivity in ctDNA detection, which significantly eliminated false-negative results in the serum or urine samples due to the low abundance of ctDNA. Such a simple-designed platform offers a user-friendly yet powerful diagnosis technique with a wide applicability, ranging from early tumor diagnosis to infection screening.
Asunto(s)
ADN Tumoral Circulante , Neoplasias , Ácidos Nucleicos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , ADN Tumoral Circulante/genética , Carcinogénesis , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
Point-of-care testing (POCT) can be the method of choice for detecting infectious pathogens; these pathogens are responsible for not only infectious diseases such as COVID-19, but also for certain types of cancers. For example, infections by human papillomavirus (HPV) or Helicobacter pylori (H. pylori) are the main cause of cervical and stomach cancers, respectively. COVID-19 and many cancers are treatable with early diagnoses using POCT. A variety of nucleic acid testing have been developed for use in resource-limited environments. However, questions like unintegrated nucleic acid extraction, open detection systems increase the risk of cross-contamination, and dependence on expensive equipment and alternating current (AC) power supply, significantly limit the application of POCT, especially for on-site testing. In this paper, a simple portable platform is reported capable of rapid sample-to-answer testing within 30 min based on recombinase polymerase amplification (RPA) at a lower temperature, to detect SARS-CoV-2 virus and H. pylori bacteria with a limit of detection as low as 4 × 102 copies mL-1 . The platform used a battery-powered portable reader for on-chip one-pot amplification and fluorescence detection, and can test for multiple (up to four) infectious pathogens simultaneously. This platform can provide an alternative method for fast and reliable on-site diagnostic testing.
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COVID-19 , Enfermedades Transmisibles , Ácidos Nucleicos , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Sistemas de Atención de PuntoRESUMEN
The outbreak of the coronavirus (COVID-19) has heightened awareness of the importance of quick and easy testing. The convenience, speed, and timely results from point-of-care testing (POCT) in all vitro diagnostic devices has drawn the strong interest of researchers. However, there are still many challenges in the development of POCT devices, such as the pretreatment of samples, detection sensitivity, specificity, and so on. It is anticipated that the unique properties of nanomaterials, e.g., their magnetic, optical, thermal, and electrically conductive features, will address the deficiencies that currently exist in POCT devices. In this review, we mainly analyze the work processes of POCT devices, especially in nucleic acid detection, and summarize how novel nanomaterials used in various aspects of POCT products can improve performance, with the ultimate aims of offering new ideas for the application of nanomaterials and the overall development of POCT devices.
RESUMEN
DNA detection plays an important role in the rapid screening of cancers and early diagnosis of infectious diseases. Here, we developed a simple, versatile, electric field-enhanced (EFE), electrochemical CRISPR biosensor to detect DNA targets in a homogeneous solution phase. To improve the detection sensitivity, we applied a pulsed electric field to enrich nucleic acids on the electrode surface. The EFE electrochemical CRISPR biosensor takes advantage of the diffusivity difference between electrochemical oligonucleotide probes and CRISPR-cleaved probes toward a negatively charged working electrode, enabling simple and sensitive electrochemical detection of DNA without the need for complicated immobilization processing of electrochemical probes. Our developed CRISPR biosensor directly detects unamplified human papillomavirus-16 (HPV-16) DNA with a sensitivity of 1 pM. Further, the EFE electrochemical CRISPR biosensor coupled with recombinase polymerase amplification (RPA) successfully detects HPV-16 DNA in clinical samples. Thus, the EFE electrochemical CRISPR biosensor provides a simple, robust, and sensitive detection method for nucleic acid-based molecular diagnostics.
Asunto(s)
Técnicas Biosensibles , Ácidos Nucleicos , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , Técnicas Electroquímicas , HumanosRESUMEN
The ability to easily produce or procure sequencing data has expanded to be within the reach of most clinics and research laboratories, but the complexity of sequence analysis remains a hurdle for many scientists, and a decline in sequencing cost means that the generation of gratuitous information in a given experiment is a challenge that is more and more often being encountered. To address this issue, methods have been present, some dating to the advent of nucleic acid sequencing, for capturing, targeting, or otherwise enriching specific nucleic acids in order to obtain greater depth of reads from a small portion of sequences within a complex sample. However, many of these methods have been complicated and laborious, relying on the design of hundreds to thousands of oligonucleotide probes, fabrication of microarray chips, and long hybridization times. Here, we review these methods, their benefits and uses, and catalog and discuss the implications of a recent development that has enabled a more efficient and expanded set of tools for enriching nucleic acids - the application of CRISPR technology. This introduction and analysis of the capabilities of new CRISPR-based enrichment strategies shows that it has the potential to expand the scope of enrichment to new possibilities, including the coupling of DNA and RNA targeting with long-read, portable sequencing platforms. Moreover, there are several areas where CRISPR-enrichment is a logical next step to more powerful and simplified sequencing for applications such as diagnostics and environmental monitoring.