Transforming Hormone Monitoring: The Emergence Of Paper-Based Analytical Platforms

Hormones are crucial biochemical messengers that regulate a myriad of physiological processes, including metabolism, growth, reproduction, and mood. Accurate detection of hormones is essential for diagnosing endocrine disorders, monitoring treatment efficacy, and ensuring overall health.

Traditional hormone detection methods, such as bioassays, immunoassays, and receptor assays, have been the cornerstone of diagnostic practices for decades. However, these methods come with several challenges. Bioassays, while providing physiological relevance, often lack specificity and sensitivity. Immunoassays, though highly sensitive, are prone to cross-reactivity and require expensive reagents and specialized equipment. Receptor assays, while offering high specificity, are limited by the availability of suitable receptors and the complexity of the assay setup.

Fig.1 Different hormones in non-invasive and invasive body fluids. (Kelkar N., et al., 2022)

The Advent of Paper-Based Analytical Devices

In response to the limitations of traditional methods, which often require expensive equipment, skilled personnel, and complex procedures, paper-based analytical devices have emerged as a truly revolutionary alternative. These innovative devices capitalize on the unique and inherent advantages of paper—its low cost, widespread availability, ease of fabrication, and seamless compatibility with a variety of printing methods—to create diagnostic tools that are not only simple and portable but also remarkably user-friendly. They have the potential to democratize diagnostic testing by making it accessible to a broader range of users, including those in resource-limited settings.

Paper-based devices operate on the sophisticated principles of microfluidics, where fluid samples are precisely manipulated within micro-scale channels and zones that are meticulously defined by hydrophobic barriers on the paper surface. This ingenious design allows for the seamless integration of multiple assay steps onto a single, compact platform. As a result, it facilitates rapid and highly efficient detection, significantly reducing the time and effort required for diagnostic processes. This advancement holds great promise for improving healthcare outcomes by enabling faster and more convenient testing options.

Mechanisms of Paper-Based Hormone Detection

Colorimetric Detection

Colorimetric assays are among the most widely used methods in paper-based devices. These assays rely on the visible color change induced by a biochemical reaction, which can be easily observed with the naked eye or quantified using a smartphone camera and image analysis software.

For instance, a study by Zangheri et al. demonstrated the detection of cortisol in saliva using a chemiluminescent lateral flow immunoassay (LFIA) integrated into a smartphone. The assay employed a competitive immunoassay format, where cortisol-peroxidase conjugates reacted with a chemiluminescent substrate to produce a detectable signal. This method achieved rapid results (<30 minutes) and a dynamic detection range of 0.3 to 60 ng/mL.

Electrochemical Detection

Electrochemical sensors offer high sensitivity and selectivity for hormone detection. These sensors typically consist of electrodes modified with specific recognition elements, such as enzymes, antibodies, or aptamers, that interact with the target hormone to produce an electrical signal.

A notable example is the development of a paper-based electrochemical biosensor for cortisol detection in human saliva by Khan et al. This sensor utilized a graphene nanoplatelet and amphiphilic diblock copolymer composite coated on filter paper, with micro-Au electrodes deposited on top. The sensor exhibited a wide detection range from 1 pg/mL to 10 ng/mL and a response time of 10 minutes.

Fluorescence-Based Detection

Fluorescence-based assays offer high sensitivity and the potential for multiplexed detection. These assays employ fluorescent dyes or quantum dots that emit light upon excitation, allowing for the quantification of target hormones.

For example, a study by Dalirirad and Steckl developed an aptamer-based lateral flow assay for cortisol detection in sweat using gold nanoparticles functionalized with cortisol-specific aptamers. The presence of cortisol caused the aptamers to desorb from the gold nanoparticles, which were then captured on a test zone, producing a visible fluorescent signal. This method achieved a dynamic range of 8 to 140 ng/mL and an analysis time of approximately 150 minutes.

Advancements in Non-Invasive Body Fluid Analysis

• Saliva-Based Detection
  Saliva has emerged as an exceptionally attractive non-invasive body fluid for hormone detection due to its ease of collection and the presence of a diverse array of biomarkers. Paper-based devices have been successfully employed to detect hormones such as cortisol, dopamine, and human chorionic gonadotropin (hCG) in saliva. These devices offer a convenient and painless alternative to traditional blood-based assays, making them particularly suitable for applications in point-of-care diagnostics and home testing.
  A study by Oh et al. demonstrated the innovative use of a trap lateral flow immunoassay (trapLFI) for cortisol detection in human saliva. This method utilized a sandwich immunoassay format with a nitrocellulose membrane and gold nanoparticles, achieving an impressive detection limit of 9.9 pg/mL and a rapid analysis time of just 15 minutes. This breakthrough highlights the potential of paper-based devices to provide highly sensitive and quick diagnostic results in a non-invasive manner.
• Sweat-Based Detection
  Sweat is another non-invasive body fluid that contains a wealth of biomarkers, including hormones. Paper-based devices have been developed to detect cortisol and dopamine in sweat, offering a painless and convenient alternative to blood-based assays. These devices leverage the natural properties of sweat to provide continuous and real-time monitoring of biomarkers, making them ideal for applications in wearable health monitoring systems.
  For instance, a lateral flow assay developed by Dalirirad and colleagues employed gold nanoparticles functionalized with cortisol-specific aptamers for cortisol detection in sweat. This assay achieved a dynamic range of 8 to 140 ng/mL and an analysis time of approximately 150 minutes. While the analysis time is relatively longer compared to saliva-based assays, the ability to monitor cortisol levels in sweat provides valuable insights into stress and metabolic conditions without the need for invasive procedures.
• Urine-Based Detection
  Urine is a readily available non-invasive body fluid that contains a variety of hormones and metabolites. Paper-based devices have been successfully applied to detect hormones such as dopamine, hCG, and testosterone in urine. These devices offer a non-invasive and cost-effective solution for hormone monitoring, particularly in settings where frequent testing is required.
  A study by Dalirirad and Steckl developed a lateral flow assay for dopamine detection in urine using DNA aptamers. This assay employed a duplex aptamer dissociation mechanism, achieving a detection limit of less than 10 ng/mL and a rapid detection time of 15 minutes. This advancement underscores the versatility of paper-based devices in detecting low-concentration biomarkers in complex biological fluids, paving the way for their broader application in clinical diagnostics and home health monitoring.

Invasive Body Fluid Analysis: Serum and Beyond

Future Trends and Challenges

• Electro-Kinetics and Hormone Aggregation
  Electro-kinetic processes are emerging as a highly promising avenue for enhancing the sensitivity of paper-based hormone detection. By applying a low-voltage direct-current field, charged particles within the sample can be precisely manipulated, leading to significant improvements in detection limits. This approach leverages the principles of electrophoresis and electroosmosis to drive analytes towards detection zones, thereby increasing the concentration of target molecules at the sensor interface.
  Recent studies have demonstrated the successful application of electro-kinetic mechanisms for protein concentration and separation on paper-based devices. These techniques hold great potential for adaptation to hormone detection, particularly for electrically neutral molecules like cortisol and estrogen. By leveraging aggregation phenomena, these molecules can be concentrated and detected more efficiently. However, challenges remain in optimizing the electro-kinetic parameters to ensure reproducibility and scalability, especially when dealing with complex biological samples that may contain interfering substances.
• Aptamer-Based Sensors
  Aptamers, which are single-stranded DNA or RNA sequences that bind specifically to target molecules, offer unparalleled specificity and affinity for hormone detection. Aptamer-based paper sensors have already been successfully developed for hormones such as 17β-estradiol and cortisol, showcasing their potential for highly sensitive and selective detection. These sensors can be easily integrated into paper-based platforms, providing a versatile and cost-effective solution for point-of-care diagnostics.
  However, several challenges must be addressed to ensure the reliability and accuracy of aptamer-based sensors. One key issue is the potential for false positives or negatives, particularly in the presence of structurally similar molecules that may bind to the aptamers. Further research is needed to optimize aptamer selection and validation processes, ensuring high specificity and stability under various environmental conditions. Additionally, the development of robust and scalable manufacturing techniques for aptamer-based sensors will be crucial for their widespread adoption in clinical and consumer applications.
• Wearable Biosensors
  Wearable biosensors represent the next frontier in hormone detection, offering continuous real-time monitoring with minimal invasiveness. These innovative devices can be seamlessly integrated into everyday items such as eyeglasses, contact lenses, textiles, or smartwatches, leveraging paper-based or other flexible substrates to detect hormones in body fluids like sweat, tears, and interstitial fluid.
  The potential applications of wearable biosensors are vast, spanning from personalized healthcare monitoring to sports analytics and beyond. They provide a unique opportunity to track physiological changes in real-time, enabling early detection of health issues and personalized interventions. However, several challenges must be overcome to realize their full potential. Ensuring the comfort and durability of these devices over extended periods of use is critical, as user compliance will depend on their seamless integration into daily life. Additionally, maintaining the accuracy and reliability of hormone detection in the presence of environmental factors and varying physiological conditions remains a significant challenge.
  Moreover, the development of efficient data processing and transmission systems will be essential for translating the raw sensor data into actionable insights. Addressing these challenges will require interdisciplinary collaboration, combining expertise in materials science, bioengineering, and data analytics to create wearable biosensors that are both effective and user-friendly.

If you have related needs, please feel free to contact us for more information or product support.

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