Coupling smartphone and <scp>CRISPR–Cas12a</scp> for digital and multiplexed <scp>nucleic acid</scp> detection

Yu, Tao; Zhang, Shengwei; Matei, Razvan; Marx, William; Beisel, Chase L.; Wei, Qingshan

doi:10.1002/aic.17365

Cited by 41 publications

(40 citation statements)

References 49 publications

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“…Second, as shown in Figure b, FOM in the order of 1–10 fM·min could be achieved within the digitalization categories with or without preamplification. This means that a target concentration of 100 aM to 1 fM could be obtained in 10 min CRISPR reaction time using digital assays without amplification, which was experimentally validated. ,, The best FOM performance was observed by combining digital assay and multiple crRNA cases, where FOM deceased to 24 aM·min . As a result, digital CRISPR assay provides the most appealing method for amplification-free CRISPR-based nucleic acid sensing.…”

Section: Performance Benchmarkingmentioning

confidence: 75%

“…Assuming that all other factors remain the same, Cas proteins with higher k cat would decrease the FOM of the CRISPR system. Different Cas proteins have shown different trans-cleavage activity with various catalytic rates. ,− Figure b presents the k cat of different CRISPR effectors reported by different groups. ,,,,− It should be noted that these results do not cover all discovered Cas proteins. Further studies are needed to explore the kinetics of various uncharacterized Cas proteins.…”

Section: Fom Improvement Strategiesmentioning

confidence: 99%

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Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

et al. 2022

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Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid-sensing systems have grown rapidly in the past few years. Nevertheless, an objective approach to benchmark the performances of different CRISPR sensing systems is lacking due to the heterogeneous experimental setup. Here, we developed a quantitative CRISPR sensing figure of merit (FOM) to compare different CRISPR methods and explore performance improvement strategies. The CRISPR sensing FOM is defined as the product of the limit of detection (LOD) and the associated CRISPR reaction time (T). A smaller FOM means that the method can detect smaller target quantities faster. We found that there is a tradeoff between the LOD of the assay and the required reaction time. With the proposed CRISPR sensing FOM, we evaluated five strategies to improve the CRISPR-based sensing: preamplification, enzymes of higher catalytic efficiency, multiple crRNAs, digitalization, and sensitive readout systems. We benchmarked the FOM performances of 57 existing studies and found that the effectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found that digitalization is the most promising amplification-free method for achieving comparable FOM performances (∼1 fM·min) as those using preamplification. The findings here would have broad implications for further optimization of the CRISPR-based sensing.

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Section: Performance Benchmarkingmentioning

confidence: 75%

Section: Fom Improvement Strategiesmentioning

confidence: 99%

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

et al. 2022

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“…These strategies have been shown to push the detection limit to nanomolar or even attomolar levels [30], which may further enhance the diagnostic power of miRNA-based liquid biopsy. Furthermore, multiplexed detection of miRNAs can be achieved by using CRISPR/Cas9 [31,32] or CRISPR/Cas12a [33] techniques, surface-enhanced Raman scattering (SERS) biosensors [34], and the biotin-neutravidin reaction [35]. Multiplexed miRNA detection may help further distinguish the origin of cancer and elevate detection specificity.…”

Section: Discussionmentioning

confidence: 99%

Cancer Detection Using an Artificial Secretable MicroRNA Found in Blood and Urine

et al. 2022

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Biomarkers can potentially help in the detection and prognosis of diseases such as cancer, its recurrence, predicting response to therapy, and monitoring of response during and/or after treatment. Endogenous tumor blood biomarkers suffer from low concentrations that are not distinguishable from background noise and, if identified, the localization of the biomarker production site is not known. The use of exogenously introduced or artificial biomarkers can eliminate these issues. In this study, we show that cancer cells can be made to produce an artificial secreted microRNA (Sec-miR) that can be detected in media from cells in culture, and from both blood and urine in living mice. In culture, we show that chaining a number of Sec-miR sequences in a plasmid and transfecting cells with the plasmids could increase Sec-miR secretion as the number of sequences increases. Tumor induction in mice with a stably transfected HeLa cell line shows the presence and significant increase in the Sec-miR with time and tumor growth in plasma (p < 0.001, R2 = 0.5542). The relative half-life of the Sec-miR was seen to be 1.2 h in the plasma of living mice and was seen to appear in urine within 12 h. The transgene for the Sec-miR within a minicircle was introduced via the tail-vein into subcutaneous tumor-bearing mice. As the tumor growth increased with time, further in vivo transfection of the Sec-miR minicircles showed an increase in Sec-miR in both plasma and urine (R2 = 0.4546). This study demonstrated that an exogenous Sec-miR biomarker would allow for early tumor detection using in vitro diagnostics techniques.

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“…Moreover, considering the advantages of 3D printing, including ease of operation, allowance for testing‐based optimization, and unique device architecture, digital microfluidic devices combined with 3D printing technology can be used for portable and ultrasensitive detection. [ 128 , 129 , 167 ]…”

Section: Clustered Regularly Interspaced Short Palindromic Repeats‐ba...mentioning

confidence: 99%

Clustered Regularly Interspaced short palindromic repeats‐Based Microfluidic System in Infectious Diseases Diagnosis: Current Status, Challenges, and Perspectives

Xie

Chen

et al. 2022

Advanced Science

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Mitigating the spread of global infectious diseases requires rapid and accurate diagnostic tools. Conventional diagnostic techniques for infectious diseases typically require sophisticated equipment and are time consuming. Emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) detection systems have shown remarkable potential as next-generation diagnostic tools to achieve rapid, sensitive, specific, and field-deployable diagnoses of infectious diseases, based on state-of-the-art microfluidic platforms. Therefore, a review of recent advances in CRISPR-based microfluidic systems for infectious diseases diagnosis is urgently required. This review highlights the mechanisms of CRISPR/Cas biosensing and cutting-edge microfluidic devices including paper, digital, and integrated wearable platforms. Strategies to simplify sample pretreatment, improve diagnostic performance, and achieve integrated detection are discussed. Current challenges and future perspectives contributing to the development of more effective CRISPR-based microfluidic diagnostic systems are also proposed.

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Coupling smartphone and CRISPR–Cas12a for digital and multiplexed nucleic acid detection

Cited by 41 publications

References 49 publications

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

Cancer Detection Using an Artificial Secretable MicroRNA Found in Blood and Urine

Clustered Regularly Interspaced short palindromic repeats‐Based Microfluidic System in Infectious Diseases Diagnosis: Current Status, Challenges, and Perspectives

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