Creating a Fluorescent Substrate for HRP Diagnostic Assays Outperforming Commercial ADHP

Fluorescence-based diagnostic assays have been an essential tool in modern medicine, particularly for detecting biomarkers and other analytes in biological samples. Among the most commonly used reagents in such assays is Horseradish Peroxidase (HRP), an enzyme that catalyzes a variety of reactions, including the oxidation of fluorescence substrates. Over the years, 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), also known as Amplex Red, has been the gold standard for HRP-based fluorescence assays. Despite its widespread use, ADHP has limitations that can compromise its efficacy in certain diagnostic applications. Recently, a novel HRP substrate, AR-2, has been introduced, offering superior performance and overcoming several of the shortcomings of ADHP.

Schematic illustration of the design principles for hydroresorufin analogues. Fig.1 Schematic graph shows the design principles of hydroresorufin analogues. (Zhou Z., et al., 2024)

Understanding the Mechanism of HRP-Based Fluorescence Assays

HRP plays a crucial role in biochemical diagnostics, particularly in enzyme-linked immunosorbent assays (ELISAs), where it is used as a reporter enzyme. When HRP reacts with hydrogen peroxide (H2O2) in the presence of a suitable substrate, it catalyzes the oxidation of the substrate, producing a fluorescent signal. This signal can be quantitatively measured, providing valuable diagnostic information.

For many years, ADHP has been the fluorescence substrate of choice for HRP-based assays due to its high sensitivity and relatively broad application. The fluorescence generated by ADHP (via its conversion to resorufin) is used to detect various biochemical markers, such as antibodies, nucleic acids, and metabolites, in clinical and research laboratories.

However, while ADHP is effective in many applications, it is not without its limitations. These include susceptibility to interference from other enzymes, instability of the fluorescent product, and sensitivity to pH fluctuations. These drawbacks have prompted the development of more reliable and robust alternatives, among which AR-2 has emerged as a promising candidate.

The Limitations of ADHP in Diagnostic Assays

Non-Specific Reactivity to Carboxylesterases

One of the key limitations of ADHP in diagnostic assays is its non-specific reactivity to carboxylesterases (CES), which are enzymes commonly found in biological samples. CES can hydrolyze the acetyl group in ADHP, triggering the production of resorufin without the involvement of HRP. This leads to false positives and compromised assay accuracy, particularly when analyzing complex biological samples such as serum or tissue extracts.

Instability of Fluorescent Products

The fluorescence produced by ADHP is also prone to instability, especially in the presence of high concentrations of H2O2 or HRP. The fluorescent product, resorufin, can undergo further oxidation, resulting in a decrease in fluorescence intensity over time. This degradation limits the sensitivity and reliability of ADHP-based assays in certain conditions, particularly when long-term monitoring or high-concentration assays are required.

pH Sensitivity

ADHP's performance is also highly sensitive to changes in pH. In acidic conditions, the fluorescence intensity of resorufin decreases rapidly, which can be problematic in assays that involve biological samples with fluctuating pH levels. The instability of ADHP under these conditions makes it less suitable for applications requiring consistent and reliable fluorescence readings across a range of pH values.

AR-2: A Superior HRP Substrate

Design and Development of AR-2

In response to the limitations of ADHP, AR-2 has been developed as a new HRP substrate with enhanced performance. The primary innovation behind AR-2 is the modification of the hydroresorufin scaffold, which underlies the fluorescence reaction. AR-2 incorporates a cyclopropylcarbonyl group at the N-position of the hydroresorufin molecule, replacing the acetyl group found in ADHP. This modification offers several advantages that make AR-2 a more reliable and efficient substrate for HRP-based assays.

Enhanced Sensitivity and Faster Reaction Time

AR-2 demonstrates superior sensitivity to HRP compared to ADHP. In kinetic studies, AR-2 exhibits a faster reaction time, reaching its peak fluorescence intensity more quickly than ADHP. This is particularly beneficial in assays that require rapid detection, as it enables quicker and more accurate measurements of HRP activity. Moreover, the fluorescence intensity of AR-2 is higher than that of ADHP under similar experimental conditions, further enhancing its sensitivity and applicability in diagnostic assays.

Resistance to Non-Specific Reactions

Unlike ADHP, which is susceptible to hydrolysis by carboxylesterases, AR-2 is designed to minimize non-specific reactivity. The cyclopropylcarbonyl group in AR-2 prevents the hydrolysis of the substrate by CES, ensuring that the fluorescence produced is solely due to the HRP-catalyzed reaction with H2O2. This makes AR-2 a more reliable substrate in complex biological samples, where CES and other interfering enzymes are present.

Stability and Tolerance to pH Fluctuations

AR-2 exhibits significantly improved stability compared to ADHP, particularly in assays that involve high concentrations of HRP or H2O2. Unlike ADHP, whose fluorescence diminishes in the presence of elevated H2O2 levels, AR-2 maintains a stable fluorescence signal even under challenging experimental conditions. This makes AR-2 ideal for assays requiring consistent performance over extended periods.

In addition, AR-2 is more tolerant to fluctuations in pH. While ADHP's fluorescence is highly sensitive to pH changes, AR-2 retains its fluorescence stability across a broad pH range, from mildly acidic to slightly basic conditions. This enhanced stability ensures more reliable results in biological assays, where pH variations are common.

Practical Applications of AR-2 in Diagnostic Assays

Uric Acid Detection

One of the primary applications of AR-2 is in the detection of uric acid (UA), a biomarker for conditions like gout and hyperuricemia. The reaction between uric acid, urate oxidase (UAO), and AR-2/HRP generates a fluorescent signal that correlates directly with UA concentration. AR-2's superior sensitivity and stability make it an ideal substrate for this application, providing more accurate and reliable results compared to ADHP.

In a typical assay, HRP is combined with AR-2 and UAO in the presence of UA. The fluorescence intensity of the reaction is measured, and the results are used to determine the concentration of uric acid in the sample. AR-2 has been shown to provide a higher signal-to-noise ratio and a more linear correlation between fluorescence intensity and UA concentration, making it a superior choice for this assay.

SARS-CoV-2 Antibody Detection

AR-2 also shows promise in immunological applications, particularly in the detection of antibodies against the SARS-CoV-2 virus. In ELISA-based assays, AR-2 can be used as the fluorescence reporter to detect IgG antibodies against the SARS-CoV-2 spike protein in human serum samples. AR-2 offers higher sensitivity and better signal quality than ADHP, which is crucial for accurate detection of low levels of antibodies in clinical samples.

In clinical testing, AR-2-based assays have been shown to distinguish between positive and negative serum samples with greater precision than ADHP-based assays. The improved sensitivity and stability of AR-2 make it an excellent choice for detecting antibodies in patients who have recovered from SARS-CoV-2 infection, as well as in diagnostic settings that require high levels of accuracy.

Fluorescence Immunoassays (ELISA)

Fluorescence immunoassays, such as ELISAs, are widely used in clinical diagnostics to detect specific antibodies, antigens, or other biomarkers in biological samples. AR-2 has been successfully incorporated into modified ELISA kits, offering enhanced sensitivity, stability, and signal-to-noise ratio compared to ADHP. The use of AR-2 in fluorescence immunoassays improves the overall performance of these tests, making them more reliable and efficient for detecting a wide range of analytes.

Advantages of AR-2 Over ADHP in ELISA Applications

Higher Sensitivity: AR-2 provides a more intense fluorescence signal, enabling the detection of lower concentrations of target analytes.
Improved Signal-to-Noise Ratio: The fluorescence produced by AR-2 is more stable and less prone to interference, resulting in clearer and more accurate results.
Better Performance in Complex Samples: AR-2's resistance to non-specific reactions with enzymes like CES makes it ideal for use in complex biological matrices, such as serum or tissue samples.

Conclusion: AR-2 as the Future of Fluorescence-Based Diagnostic Assays

The introduction of AR-2 marks a significant advancement in the field of HRP-based diagnostic assays. With its superior sensitivity, resistance to non-specific reactivity, enhanced stability, and tolerance to pH fluctuations, AR-2 addresses the major limitations of ADHP and sets a new standard for fluorescence-based diagnostics. From uric acid detection to SARS-CoV-2 antibody testing, AR-2 is proving to be a versatile and reliable substrate for a wide range of diagnostic applications.

As diagnostic technologies continue to evolve, AR-2 is poised to become the preferred choice for researchers and clinicians seeking more accurate, reliable, and efficient fluorescence assays. With its improved performance and robust features, AR-2 is positioned to play a crucial role in the future of in vitro diagnostics, paving the way for more precise and accessible healthcare solutions.

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Reference

Zhou, Zhichao, et al. "Developing a fluorescence substrate for HRP-based diagnostic assays with superiorities over the commercial ADHP." Chinese Chemical Letters 35.6 (2024): 108970.

This article is for research use only. Do not use in any diagnostic or therapeutic application.