A versatile CRISPR/Cas12a-based sensitivity amplifier suitable for commercial HRP-based ELISA kits
Introduction
Advances in CRISPR/Cas biosensing have led to a successful development of diverse detection systems for nucleic acids [[1], [2], [3]], proteins, small molecules, etc [2,[4], [5], [6], [7], [8], [9]]. A special group of Cas effectors such as Cas12 and Cas13 with collateral cleavage activity, and highly specific target binding capability [1,[10], [11], [12]], provide programable signal amplification, and they have been widely explored for the development of various biosensing schemes [1,2,[13], [14], [15]]. In this approach, the Cas effectors are activated by the target nucleic acid sequence complementary to the guide RNA, and, once in this activated conformation, they are able to randomly degrade the surrounding nucleic acids [[13], [14], [15]]. By introducing nucleic acid-linked fluorescence-quenched probes, this unique and highly efficient sequence-independent degradation activity results in an amplified fluorescence signal [3,[16], [17], [18]]. The superior sensitivity, specificity and simplicity of CRISPR/Cas-based biosensing [15,17,18] combined with its versatility to integrate with other biosensing components, such as allosteric transcription factors (aTFs) [6], aptamers [4,7,19], nanoparticles [9] or antibodies [5], offer diverse opportunities to establish novel biosensors for beyond nucleic acid detection [1,18,[20], [21], [22]].
Enzyme-linked immunosorbent assays (ELISA), which relies on the specific antibody-antigen interaction for target analyte recognition [23], are among the leading analytical approaches for the detection of non-nucleic acid targets. Such assays are widely applied in research and clinical diagnostics due to their specificity, simplicity and reliability [[23], [24], [25], [26]]. However, the key performance measures for such ELISA systems, the sensitivity and detection range tend to be limited, and, in some cases, they may be insufficient. It may also be impossible to select a single ELISA kit with a suitable combination of sensitivity and detection range, particularly for analyte with wide physiological concentrations such as cytokines IFN-γ whose concentration in the living organisms can greatly vary from sub-pg/mL level to ng/mL level under different conditions [27,28]. To address the sensitivity issue, diverse approaches have been proposed, including engineering the ELISA plates for improved capture antibody density and orientation [23,[29], [30], [31]], increasing detection antibody or enzyme load with nanoparticles or polymers [32,33], boosting enzyme efficiency with enzyme amplifiers [[34], [35], [36]], multiple enzymatic amplification steps [37], and replacing the signal amplification strategies with polymerase amplification [28] or DNAzymes [38], etc. Additionally, DNA-based techniques with DNA-modified antibodies have also been used to introduce nucleic acid synthesis into immunoassays, such as Polymerase Chain Reaction (PCR) [39], Recombinase Polymerase Amplification (RPA) [40], Loop-mediated isothermal amplification (LAMP) [41] and Rolling Circle Replication (RCA) [42], for signal amplification. These modifications are mainly system-specific, allowing different manufacturers to build ELISA kits with greatly varying performance, even for the same analyte. They can not be immediately transferred from one immunoassay to another, or directly applied to established ELISA platforms without significant adaptations. A simple and universal strategy to increase the sensitivity of commercial ELISA kits can be an efficient and a cost-effective approach to overcome the above problem of poor universality. However, the details of reagents and buffers in a commercial ELISA kit are generally undisclosed, and can vary significantly between different manufacturers. This presents a major obstacle for integrating additional component into established ELISA kits, where the potentially negative interaction between unknown reagents from the ELISA kit and the newly applied components can jeopardise the performance of the entire system.
This work reports how to use the CRISPR biosensing approach to enhance the sensitivity of commercial ELISA kits which use HRP but have otherwise unknown chemistry. Our strategy transfers the CRISPR/Cas12a collateral cleavage function into an independent signal amplification module hence we termed it CRISPR/Cas12a-based ELISA Sensitivity Amplifier (CES-Amplifier). The central component is a specifically designed conjugate of a short single strand DNA (ssDNA) and anti-HRP antibody. This conjugate is able to activate CRISPR/Cas12a without compromising its activation efficiency for the conjugated ssDNA, while the affinity for the conjugated antibody is also largely unaffected. By using the anti-HRP antibody, our CES-Amplifier has been directly applied to a commercial ELISA kit for IFN-γ detection without modifying its original reagents or protocol. Comparing to its original ELISA assay performance, applying the CES-Amplifier resulted in over two orders of magnitude of sensitivity increase from the original sensitivity of 312.5 pg/mL to 1.2 pg/mL, along with 1 order of magnitude increase in detection range. This is important e.g. for comprehensive evaluation of IFN-γ changes under certain physiological conditions such as in blood where IFN-γ levels can vary from 17 pg/mL to 1500 pg/mL [27,28], which is below the originally claimed detection limits of the commercial ELISA kits [28,43,44]. More importantly, without the need for full control of ingredients in unknown immunoassays, but, instead, by simply replacing the original kit buffer system with our own controlled reaction environment after one additional washing, our anti-HRP-based CES-Amplifier approach has the potential to be directly applied, as a stand-alone addition, to a wide range of other commercial ELISA kits which use the HRP-conjugated antibody. Under such circumstances, our CES-Amplifier provides a deliverable, user-friendly and affordable biosensing innovation for the end-users, and also represents a significant advance towards further integration of the CRISPR/Cas technology into the mainstream biosensing applications for the end-users.
Section snippets
Materials
Phosphate buffered saline (PBS) (Sigma, 10 mM, pH = 7.4), Lightning-Link Streptavidin Conjugation Kit (Abcam, ab102921), streptavidin (Sigma), recombinant human IFN-γ protein (R&D, 285-IF-100), Donkey Anti-Mouse IgG (H + L) Affinity Purified PAb (B&D, D201CABS2), Donkey Anti-Rabbit IgG (H + L) Affinity Purified PAb (B&D, D301CABS2), Human IFN gamma ELISA Kit (Abcam, ab174443), EnGen® Lba Cas12a (Cpf1) protein (New England Biolab), 10X NEB 2.1 buffer (New England Biolab), bovine serum albumin
Schematics of the CRISPR/Cas12a-based ELISA sensitivity amplifier
Bringing together the highly efficient collateral cleavage activity of CRISPR/Cas12a with the conventional sandwich immunoassay format forms the central idea in the CES-Amplifier. This has been realized by the synthesis of a single strand DNA (ssDNA) oligo and anti-HRP antibody (ssDNA-Abs) conjugate. This bi-functional ssDNA-Abs conjugate needs to retain sufficient specific target recognition function as the original antibody, and it also must play a role of the nucleic acid target for the
Discussion and conclusions
The collateral cleavage activity in certain Cas effectors such as Cas12a, is a central feature of CRISPR/Cas systems to realize rapid and sensitive biomolecular detection [1,13,14,18]. An efficient fluorescence signal amplification scheme based on collateral cleavage has been integrated into various types of biosensing systems, including for non-nucleic acid detection [[4], [5], [6], [7], [8]]. This can be achieved by using a short DNA fragment either acting as the analyte recognition molecule,
CRediT authorship contribution statement
Yi Li: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Data curation, Visualization. Fei Deng: Methodology, Investigation, Validation, Visualization. Ewa M. Goldys: Resources, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was partially supported by the Australian Research Council Centre of Excellence for Nanoscale BiophotonicsCE14010003. Yi Li and Fei Deng acknowledge the PhD scholarship support from University of New South Wales. The protein-DNA conjugation used here is also used in our unpublished work by Ruiting Lan, Ximing Du, J. Justin Gooding, Guozhen Liu and the present authors.
Yi Li is a Research Associate in Graduate School of Biomedical Engineering, University of New South Wales (UNSW). His research interests include nuclease-associated biosensor development, deployable detection strategy and point-of-care diagnostics for cancer biomarker or pathogen detection.
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Cited by (0)
Yi Li is a Research Associate in Graduate School of Biomedical Engineering, University of New South Wales (UNSW). His research interests include nuclease-associated biosensor development, deployable detection strategy and point-of-care diagnostics for cancer biomarker or pathogen detection.
Dr. Fei Deng is a Research Associate in Graduate School of Biomedical Engineering, University of New South Wales (UNSW). His research interests include CRISPR biosensing, molecular imprinted polymer, immunosensors and deployable in vivo devices for detection of small molecules and small proteins.
Professor Ewa M. Goldys, the Deputy Director of the Australian Research Council Centre of Excellence for Nanoscale Biophotonics(CNBP), is working at Graduate School of Biomedical Engineering, UNSW. Her research focuses on the development and applications of advanced fluorescence techniques to biomedicine, nanotechnology and advanced materials. She is holding the Fellowship of the Australian Academy of Technology and Engineering while being the fellow of the International Optical Society.
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These authors contributed equally to this work.