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Predicting detection limits of enzyme-linked immunosorbent assay (ELISA) and bioanalytical techniques in general Shiyun Zhang,ac Alexa Garcia-D'Angeli,a Joseph P. Brennand and Qun Huo*abc The detection limit is one of the most important performance parameters for bioanalytical techniques. Here we present a generic method to estimate the detection limit of biomolecular assays based on a step-bystep analysis of the assay procedure. Enzyme-linked immunosorbent assay (ELISA) is used here as an
Received 26th September 2013 Accepted 31st October 2013
example; however, much of the information presented in this article may be applied to other types of biomolecular assays and analytical techniques. A clear understanding of what affects the detection limit
DOI: 10.1039/c3an01835k
can help researchers to evaluate different bio-analytical techniques properly, and to design better
www.rsc.org/analyst
strategies to optimize and achieve the best analytical performance.
Introduction The detection limit is of great importance in biomolecular assay and sensor development. There has been an increasing pressure to push the detection limit of bioanalytical techniques to lower levels. This pressure is largely driven by our demand for new molecular diagnostic tests for early stage cancer detection and diagnosis.1,2 At early stages of cancer development, the amount of cancer biomarker molecules released from the tumor to the blood or other biological uids is very small.3,4 Naturally, one assumes that a more sensitive analytical technique that can “catch” these cancer biomarkers at lower concentrations will allow cancer to be detected earlier. Under this general premise, pursuing lower detection limits has become a major goal of new bioanalytical technology development. It is not uncommon to see detection limits in the fg–pg mL1 range for protein analytes, and sometimes even down to the single molecule level.5–10 Table 1 provides some representative examples of detection limit claims of commercial products and some newer assays that are under development. While pursuing bioanalytical techniques and products with higher sensitivities and lower detection limits, it is important to ask a critical question: is the claimed/expected detection limit theoretically achievable? If by theory, an analytical method cannot possibly achieve the sensitivity as claimed, attempted use of such methods for expected high sensitivity analysis can
a
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA. E-mail:
[email protected]; Fax: +1 407-882-2819; Tel: +1 407-882-2845
b
Department of Chemistry, University of Central Florida, Orlando, FL 32826, USA
c
Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL 32826, USA d
Department of Mathematics, University of Central Florida, Orlando, FL, USA. Tel: +1 407-882-2710
This journal is © The Royal Society of Chemistry 2014
only lead to a waste of research effort and resources, and sometimes, misleading results. In 2007, a report by Getzenberg et al. claiming the discovery of a new prostate cancer biomarker, early prostate cancer antigen-2 (EPCA-2), initially attracted enormous publicity.11 However, doubts were raised on this work by other researchers based on the fact that the assay used by the authors cannot possibly achieve the detection limit as claimed in the report.12 Later this paper was retracted because the nding could not be duplicated. If more careful attention were paid to the sensitivity of the assay used in the study, this kind of mistake and the subsequent damage caused to the research community could have easily been avoided. For commonly used bioanalytical techniques and assays, the detection limits have been well established and a large amount of data is available for comparison. However, in recent years, an enormous number of new bioanalytical assays and sensors have been developed and reported, especially with the rise of nanotechnology-based platforms.13–17 Without a substantial amount of data from other users to support the claims made by the researchers and developers, it is difficult to know the realistic detection limit of the new technique and to compare the new technique with existing techniques. A better understanding of every step of the assay and the theoretical detection limit that a new assay can possibly achieve is not only important for comparison purpose, but also can help researchers and developers to design better strategies to optimize and achieve the best sensitivity of the technique. Driven by these needs, we feel it is necessary to re-visit some of the fundamental aspects of biomolecular assays. We present here a generic procedure that may be used to estimate the theoretical detection limit that a bioassay may achieve. The enzyme-linked immunosorbent assay (ELISA) is one of the most extensively used assay techniques for biomolecular detection and analysis, especially for protein analytes. There are
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Table 1
Paper
Detection limit claims of some representative commercial ELISA kits and nanotechnology-enabled new assays
Detection method
Company/reference Product name
Limit of detection
Assay range
Detection method/notes
Colorimetric
Life Technologies
2 pg mL1 0.4 ng mL1