Dual-amplified CRISPR-Cas12a bioassay for HIV-related nucleic acids

Zhou, Jing; Hu, Jianyu; Liu, Rui; Wang, Chaoqun; Lv, Yi

doi:10.1039/d2cc00792d

Cited by 19 publications

(18 citation statements)

References 36 publications

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“…After magnetic separation, the number of remaining PtNP tags in the supernatant was directly counted by sp-ICP-MS, exhibiting a considerable correlation against CEA. In accordance with references from other leading research groups and our previous studies, 11,49,50 the proposed method could also be utilized in nucleic acid binding or assay research and other types of interface reactions. We also carried out a size (diameter) optimization of nanoparticles on the binding efficiency and discussed the results, screening the suitable size of PtNPs (34 nm) with high efficiency for differential immunoassay.…”

Section: ■ Conclusionsupporting

confidence: 79%

Single-Nanoparticle Differential Immunoassay for Multiplexed Gastric Cancer Biomarker Monitoring

Huang

Zhao

et al. 2022

Anal. Chem.

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Precision medicine demands the best application of multiple unambiguous biomarkers to bring uniform decisions in disease prognosis. The remarkable development of heterogeneous immunoassay greatly promotes precision medicine when combined with the biomarker combination strategy. Nevertheless, the cumbersome washing steps in heterogeneous immunoassay have inevitably compromised the accuracy because of the sample losses and nature change of the matrix, challenging the further exploration of a more facile and lower limit-of-detection analysis. The new methodologies with high throughputs and specificity are never out of date to provide simultaneous evaluations and uniform decisions on multiple analytes through a simple process. Herein, we propose a new wash-free immunoassay, named differential assay, for multiplexed biomarker monitoring. The method is based on counting the number difference of unbound nanoparticle tags before and after immunoreactions from a solid support (i.e., magnetic microsphere) by single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS), discarding the tedious washing steps. We primarily explore the proof-of-concept proposal within two types (sandwich and competitive assay), demonstrating the good feasibility for further facile clinical practice. To provide efficient multiplexed evaluations, we synthesized PtNPs with four diameters and screened the most suitable size for efficient differential immunoassay. The wash-free strategy was successfully utilized in simultaneous serological biomarker (CA724, CA199, and CEA) evaluation, with results in good accordance with those measured by the clinical routine method. Potentially, the proposed differential bioassay can be regarded as a more facile and valuable tool in malignancy prognosis and cancer recurrence monitoring.

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Section: ■ Conclusionsupporting

confidence: 79%

Single-Nanoparticle Differential Immunoassay for Multiplexed Gastric Cancer Biomarker Monitoring

Huang

Zhao

et al. 2022

Anal. Chem.

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“…Lv et al developed a CRISPR-Cas12a-based assay for the detection of HIV-related DNA using the ICP-MS signal of AuNPs (Figure 9A). [108] In this work, the 3'-end of the probe bound to AuNPs and the 5'-end was connected with magnetic beads. When the target was present, the trans-cleavage activity of the CRISPR-Cas12a system was activated to cleave AuNPstagged probes, leading to the release of AuNPs from the surface of magnetic beads.…”

Section: Other Biosensorsmentioning

confidence: 99%

Section: Cas12a‐based Biosensors Using Gold Nanomaterialsmentioning

confidence: 99%

Gold Nanomaterials‐Implemented CRISPR‐Cas Systems for Biosensing

Xianyu

2023

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prokaryotes, [1] which acts as an immune weapon in bacteria to eliminate virus invading. CRISPR-Cas systems developed by bacteria can effectively remove virus genes integrated into bacterial genes. [2,3] The working principle is that under the guidance of RNA, the CRISPR-Cas protein can target the nucleic acid sequence and eliminate it. [4] Inspired by CRISPR-Cas systems, researchers have adapted them to develop gene-editing techniques that have quickly become the most popular technology in life science. [5,6] In addition to the outstanding gene-editing capabilities, CRISPR-Cas systems show great potential as the next-generation techniques for rapid and sensitive biosensing. CRISPR-Cas systems can be easily integrated with a range of nucleic acid-based signal amplification approaches and signal probes because of the precise targeting and cleavage functions toward nucleic acid sequences. Owing to their superior properties including high specificity for identifying single-nucleotide variations, ultrahigh sensitivity, and ease of fabrication, CRISPR-Cas systems can serve as a facile and efficient tool to develop biosensors for point-of-care detections. At present, CRISPR-Cas systems are grouped into class 1 and class 2 systems that are further subdivided into different types and subtypes. Class 1 CRISPR-Cas systems (type I, III, and IV systems) use a wide assortment of smaller Cas proteins (Cas1, Cas2, Cas3, and so on) to form a multisubunit interference complex. On the contrary, class 2 systems (type II, V, and VI systems) use a single, comparatively larger Cas effector protein (Cas9, Cas12a, Cas13a, and so on) for interference and, in certain cases, CRISPR RNA (crRNA) biogenesis. [7] Notably, Cas proteins in class 2 systems have been explored to design CRISPR-Cas-based diagnostic tools, including CRISPR/Cas9-triggered isothermal exponential amplification reaction (CAS-EXPAR), [8] one-hour low-cost multipurpose highly efficient system (HOLMES), [9] HOLMES version 2 (HOLMESv2), [10] specific high-sensitivity enzymatic reporter unlocking (SHERLOCK), [11] SHERLOCK version 2 (SHERLOCKv2), [12] DNA endonuclease-targeted CRISPR transreporter (DETECTR), [13] and so on.Traditional biosensors based on CRISPR-Cas systems generally rely on fluorescent single-stranded DNA (ssDNA) for the readout, while these probes are limited by the low stability and sensitivity in complex samples. To meet these challenges, Due to their superiority in the simple design and precise targeting, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have attracted significant interest for biosensing. On the one hand, CRISPR-Cas systems have the capacity to precisely recognize and cleave specific DNA and RNA sequences. On the other hand, CRISPR-Cas systems such as orthologs of Cas9, Cas12, and Cas13 exhibit cis-cleavage or trans-cleavage activities after recognizing the target sequence. Owing to the cleavage activities, CRISPR-Cas systems can be designed for biosensing by degrading tagged nucleic acids to produce detectable...

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“…Since the discovery of Cas12a, the trans-cleavage reaction substrate, which is labeled with a fluorophore and quencher, has been designed to be 5–7 nucleotides (nt) in length to achieve better response efficiency. − However, the length of the substrate has not been thoroughly explored. Only a few studies have focused on optimizing the reaction conditions of the Cas12a detection system. − Ma et al found that divalent metal ions in the buffer affected Cas12a’s activity and that manganese ions can enhance the signal up to 13-fold compared to magnesium ions . Rossetti et al used a hairpin reporter instead of a linear reporter, which reduced the detection limit by about 15 times .…”

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confidence: 99%

“…Rossetti et al used a hairpin reporter instead of a linear reporter, which reduced the detection limit by about 15 times . Some researchers have attempted to label the reporter on gold nanoparticles, which can also reduce the detection limit by about 20 times. , Additionally, some complex designs, such as modifying the relevant sequences or using a non-fluorescent reporter system, have also been proposed to improve detection results. − However, the improvements achieved by these optimization methods are not significant, and some methods are complicated. Moreover, there is a lack of thorough investigation into the optimization of reaction conditions for the CRISPR-Cas12a system, such as the Cas12a/crRNA ratio, buffer composition, and substrate structure.…”

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confidence: 99%