Lateral Flow Immunoassays for Point-of-Care Diagnostic Testing - Richard Hoff, B.A. M.S. in Bioengineering
The topic of this week's blog post is taking a different spin on things after a while. My brother, Richard "Richie" Hoff (he/him/his), has a Bachelor's Degree and Master's Degree in Bio-Engineering from Temple University. Richie also has another Master's Degree in Biomedical Engineering from Cornell University. One of the courses that he was required to take towards his Master's Degree at Cornell was a course called Core Concepts of Disease. For one of the assignments for this course, each of the students enrolled in the class had to write individual research papers about emerging research related to disease; so, Richie chose to write his research essay about lateral flow assays. Since this topic is scientific and very informative when it comes to diagnostic testing of medical health concerns, Richie and I have agreed together that it would be beneficial to be uploaded as a guest post.
CONTENT WARNING: I would like to mention that this blog post is written for educational purposes only and not intended to provide any professional services. Social media should never be used as a substitute for medical nor mental health care. If you suspect that you and/or a loved one are experiencing any medical health concerns, it is okay to get help and I highly recommend seeking professional help from a medical doctor who is qualified to do so.
1. Lateral Flow Assays: Purpose, Significance, and Function as a Point-of-Care Diagnostic
Point-of-care (PoC) diagnostic testing offers the distinct advantage of providing portable, low-cost, rapid, and accurate in-vitro detection of biomarkers associated with disease; they allow disease diagnosis and medical decisions regarding disease management and monitoring to be performed at early stages of disease development and facilitate improved health outcomes for patients [1-3]. The global PoC diagnostics market is expected to grow to USD 50.6 billion in 2025 from USD 29.5 billion in 2020 at the compound annual growth rate (CAGR) of 11.4% [4]. Demand for diagnostic kits has significantly increased due to the COVID-19 pandemic, in addition to increased prevalence of infectious diseases throughout the world and especially in developing countries; another factor is the increasing burden of non-communicable diseases in contributing to global mortality and their disproportionate effect on low- and middle-income countries [3-7]. One such PoC diagnostic approach that has contributed to the very idea of "personalized medicine" is the lateral flow immunoassay (LFIA), seen as the original gold standard of PoC diagnostic approaches and a subtype of lateral flow assay (LFA), which originated in the late 1970s [8, 9]. The global LFA market is expected to grow to USD 10.2 billion in 2025 from USD 8.2 billion in 2020 at the CAGR of 4.5%; LFAs are dominating as the most common commercially available format of PoC diagnostic test at this time, with demand increasing for the same reasons driving the growth of the overall PoC diagnostics market [1, 4, 10].
LFAs can be utilized to detect diagnostic biomarkers associated with a diverse set of conditions including vitamin and mineral deficiencies, non-communicable diseases such as cardiovascular disease and cancer, and infectious diseases such as HIV, malaria, and tuberculosis [1, 2, 5, 8, 11]. LFA functionality relies on flow migration and flow technology, with the components of a LFA device being capable of transporting fluid via capillary action, not requiring external pumps [1, 8, 12]. The components of a typical sandwich-type LFIA include a sample pad, a conjugation pad, a membrane, and an absorbent pad that each perform specific functions in the device. These layers of the device are typically mounted onto an adhesive backing material to provide structural support that makes the device easy to handle and strengthens the fragile membranes [1, 5, 8, 12, 14-16]. Adjacent pads are overlapped to ensure consistent fluid flow between the layers of the device from the sample pad to the absorbent pad [11, 12, 15-17].
[Image Description: A lateral flow immunoassay's typical configuration. Left to right: A sample pad, conjugate release pad, membrane, test line, control line, backing card, and adsorbent pad; "Flow direction" black text center-aligned above a white arrow outline in black and pointing to the right is also above the sample pad.]
The function of a LFA device proceeds via capillary action-driven fluid transport, with the sample first being added to the adsorbent sample pad to be pre-filtered, upon which it uniformly and continuously migrates into the conjugate pad [8, 12, 13]. The sample may need to be pre-treated through methods such as incubation at a higher temperature to ensure that the target analyte interacts efficiently with the dried recognition element in the conjugate pad to create a complex. Washing buffer may also need to be added to facilitate the flow of the sample through the full length of the membrane after passing through the conjugate pad [5, 8, 17, 18]. The conjugate pad material is highly significant when it comes to the shelf-life of LFA devices, as it ensures the stability of the recognition elements and conjugates loaded inside it and is responsible for releasing them into the membrane reaction matrix [8, 12]. The conjugate pad material also has a higher capacity for pulling and holding fluid compared to the sample pad, improving flow and movement to the membrane from the sample pad [17]. The sandwich format of LFA is designed to detect a single analyte will normally have two reaction sites along its membrane [1, 8, 9, 12, 19]. The first reaction site that the mobile phase passes over along the membrane is the test line, which targets and immobilizes the analyte being detected; the second reaction site is the control line, which is an internal control utilized to ensure that the device is operating correctly [5, 8, 9, 11, 12]. These test and control lines are dispensed onto the membrane during assembly [1, 8, 9, 11, 12, 18]. The competitive format of LFA involves conjugated particles with the ability to be immobilized at both reaction sites, with the analyte competing for binding sites at the test line and not aggregating at the control line; if the sample is negative for the analyte, the conjugate particles simply bind at both reaction zones [8, 16, 19]. The absorbent pad, also referred to as a sink, wick, or waste pad, is at the end of the strip. This final pad has the purpose of maintaining the flow of reagents out of the membrane, preventing backflow of reagents that would create false positives in the results [8, 12, 16].
2. LFA Parameter Optimization
There are several parameters to consider when designing and manufacturing a functional and well-performing LFA, which makes optimization important for minimizing the time of detection; this ensures reproducible results between individual and batches of test strips, and maximizing the device's sensitivity and specificity [1, 5, 6, 8, 17, 20]. Alongside the reader technology and manufacturing process for constructing the strip, the construction of the strip and specific assay technology are the most significant parameters that must be optimized for each LFIA platform [1, 5, 14, 21]. First, a biomarker associated with the disease must be identified and selected as the particular diagnostic target, making it the analyte that will be detected in biological samples of disease- or condition-positive patients [1, 8, 12]. Human cells, nucleic acids, proteins, and microbes are among the existing diagnostic targets utilized in these assays. Samples taken from patients, which may need additional post-processing before being run through the assay, include tissue samples and bodily fluids such as saliva, blood, plasma, and urine [1, 2, 8, 12]. When preparing more quantitative diagnostic tests or those utilizing visual detection, LFAs' parameters are optimized with the intention of ensuring that the clinical range of the target biomarker(s) in the body associated with the particular condition or disease can be detected using the test. Spiked buffers containing different known concentrations of the biomarkers across their specific clinical range that act as mock clinical samples are utilized for tests conducted during the optimization process and initial optimized LFA validation. Later validation tests of the device will use real biological samples acquired directly from patients experiencing the target condition or disease being diagnosed as a step towards clinical translation [11, 15, 18, 20, 22].
An example of a well-optimized rapid LFIA was developed by the Erickson Lab at Cornell University. It was a strip designed to simultaneously detect and quantify iron (ferritin), vitamin A (retinol-binding protein), and inflammation (C-reactive protein) status for diagnosis of vitamin A and iron deficiency; sensitivities of 88%, 100%, and 80% and specificities of 97%, 100%, and 97% were respectively reported for each of these three biomarkers. These translates to below 15 ng/mL (32 pmol/L) for ferritin, below 14.7 ug/mL (0.70 ug/mL) for retinol-binding protein, and above 3.0 ug/mL (120 nmol/L) for C-reactive protein; these biomarkers' limit of detection values were all below the adult and child diagnostic threshold. An incubation pad and mixing pad were utilized in place of a standard conjugation pad to simplify the assay's operation. The TIDBIT fluorescence imaging system also designed by the Erickson Lab acquires images after 15 minutes and can be connected to their proprietary NutriPhone technology or a standard laptop to interpret and display the results. This PoC diagnostic platform has high suitability for resource-rich and resource-limited settings. The TIDBIT reader can be manufactured at a minimum of $95 using commercially available products, and since test strip materials and reagents can be bought in bulk and used to manufacture thousands of strips, each multiplex test for these three biomarkers only costs around $1.50 [11].
[Image Description: Iron and vitamin A deficiency LFIA schematic and sample fluorescence image of results. (A) Diagnostic test schematic of the multiplexed test strip for diagnosing iron and vitamin A deficiency. A sandwich lateral flow assay was used for ferritin and c-reactive protein and a competitive assay was used for retinol-binding protein; use of a sandwich or competitive assay respectively results in higher and lower fluorescence signal intensity with increasing concentration. (B) Sample results from patients with known biomarker concentrations in serum [11].]
2A. Lateral Flow Test Strip Materials
The materials for the sample pad, conjugate pad, membrane, and absorbent pad must be chosen to optimize the pads' functionality so that the test strip will exhibit high sensitivity when running samples from patients that are positive for the disease-associated biomarker [8, 17]. Cross-linked silica, rayon, cellulose, and glass fiber are commonly used as materials for sample pads, while absorbent pads are often composed of high-density cellulose [18]. Polyesters or glass fibers that have been pre-treated with surfactants, polymers, or proteins to become hydrophilic are utilized as conjugate pad materials, ensuring long-term stability and optimum conjugate release [8, 12]. The selection of the membrane material is just as important as that of the probe and recognition element with regards to determining the assay's functionality, which is due to the role of its dispensed reactive zones in capturing the analyte; LFA tests tend to utilize hydrophobic nitrocellulose, glass fiber, or cellulose acetate membranes [8, 9, 12]. Porous nitrocellulose membranes are the most widely accepted materials that have been utilized for LFA test strips; this is due to their higher protein binding properties, ease of handling and wetting, low cost, great variety and availability commercially, true capillary flow characteristics, and lack of interference with the assay [1, 8, 9, 12, 14]. The pore size of nitrocellulose membranes is an important parameter for optimizing LFA sensitivity due to its effect on capillary flow. Smaller pores reduce the speed of the assay and the time it takes for the analyte-antibody-label complex to reach and pass the test and control lines, which facilitates long reaction time and higher sensitivity, however, non-specific binding reactions also have an increased probability of occurring with decreasing assay speed. This means that a pore size that optimizes device sensitivity, balances reactive time, and minimizes non-specific binding reactions must be chosen to have a viable membrane for a LFA device [5].
2B. Biorecognition Elements
Next, a capture probe can be selected that targets the specific analyte with high affinity, as this probe regulates the assay's specificity; while aptamers, affimers, and other binders are being investigated as potential capture probes and there are different alternatives used currently, antibodies are the most well-established biorecognition elements that are commonly utilized in LFAs [1, 8, 9, 12, 23]. For this reason, LFAs that exclusively use antibodies for this purpose referred to as lateral flow immunoassays (LFIAs); there are also nucleic acid lateral flow assays (NALFAs), which detect amplicons that are able to form during the polymerase chain reaction [13, 24]. More attention in this blog post will be given to the former. Monoclonal and polyclonal antibodies are typically used individually or in combination for these assays, with each having their own advantages, and recombinant antibodies with tunable specificity are also being investigated as a potential biorecognition element for LFIAs [8]. A LFIA designed to detect a single analyte typically utilizes 2-3 types of antibodies, which are separately incorporated into the conjugate pad and immobilized on the membrane portion of the strip to generate the two reaction sites [8, 14]. The amount of antibodies in the reaction sites along the membrane also must be optimized to ensure that the maximum number of interactions between the capture antibodies on the test line and the detection antibodies bound to the analyte are occurring while remaining cost-effective [5, 6, 17]. The antibodies pre-loaded into the conjugate pad must be conjugated with a detection label to allow qualitative or quantitative detection of the analyte using the assay; these labels include fluorescent tags, latex beads, quantum dots, colloidal carbon, and gold nanoparticles [1, 8, 11, 17, 18]. Most LFAs use visual detection, whereby qualitative or semi-quantitative testing is achieved via visual examination of a color change at the test and control lines; the detection labels typically incorporated in assays relying on visual detection are gold nanoparticles or latex beads [8]. True quantitative testing that requires the use of a fluorescence or optical strip reader is possible when detection labels such as colloidal gold, paramagnetic particles, and fluorescent dyes are utilized [8]. Due to their high signal-to-noise ratios, fluorescent nanoparticles and quantum dots are perceived as more viable options for increasing LFIA sensitivity [5]. The amount of antibodies are pre-loaded into the conjugate pad must be optimized as well to maximize the amount of analyte being recognized by the detection antibodies: this promotes a greater number of interactions at the test line and ensuring a better signal [5, 6]. The optimization process facilitates screening of the antibodies' and other assay reagents' stability under assay conditions and different environmental conditions [19].
2C. Lateral Flow Immunoassay Buffer Composition and Volumes
The compositions of the running buffer and conjugate buffer, the volume of running buffer added to facilitate smooth fluid flow, and the volume of sample being tested for analyte using the assay are also significant parameters related to the functionality of the LFIA device [5, 6, 8, 14]. As previously stated, the conjugate buffer is necessary for pretreating the conjugate pad to enhance its hydrophilicity, which ensures optimal release and stability of the conjugate [6, 8]. While an additional volume of running buffer may be added to ensure that the sample can travel the length of the nitrocellulose membrane, the sample volume is significant because of its role in influencing what is known as the volume ratio [5]. While the results of the majority of LFIAs rely on visual detection, true quantitative testing is also possible [5, 8, 9, 11, 12, 14, 17, 18]. When using a quantitative LFIA to detect for the presence of an analyte, the reader technology measures and collects the fluorescence signals generated at the test and control lines; the color intensities obtained for the control and test lines can then be used to calculate the T/C ratio, also known as the volume ratio [1, 5, 8, 26]. Measuring the T/C ratio for each assay and comparing these values between individual and batches of test strips is preferable as a metric for assessing the assay's precision and reproducibility compared to simplify reading the absolute fluorescence values at the test and control lines [26]. The sample volume must provide a sufficient amount of analyte to generate analyte-antibody interactions when the sample migrates into the conjugate pad; this increases the number of conjugates that are ultimately formed and detected at the test line after the assay has reached completion as well as the resulting T/C ratio [5, 14]. The higher the sample volume, the closer in concentration the analyte in the sample will be to that of the antibody within the conjugate pad, increasing the result T/C ratio; when the sample volume and therefore the amount of analyte becomes sufficiently high, most of the large number of analyte-antibody complexes generated within the conjugate pad will likely pass through the test line without even interacting with the immobilized captured antibodies, at which point no significant change in the T/C ratio should be observed [5]. Variations in the sample volume will therefore lead to poor precision [8]. It has also been observed that as the velocity of the membrane increases, the test's required volume of sample also increases [14].
Alongside the type of nitrocellulose membrane utilized for an assay, the composition of the running buffer is also necessary for controlling the capillary flow [1, 5, 8, 14]. Slower capillary movement in the LFIA membrane increases the migration time, which can improve the detection range sensitivity by means of promoting a higher degree of inhibition. Optimization is important because an assay time that is too great can contribute to reducing sensitivity [5]. A running buffer typically contains multiple components, which each contribute to assay success, though the main component is especially important. The main component of the running buffer is responsible for rehydrating the biorecognition element in the conjugate pad and ensuring its movement into the membrane portion of the test strip. This prevents any reporter dye nonspecific binding to the pad, and achieving an optimum flow rate that makes the test rapid while also ensuring that sufficient interactions occur [5, 8]. Flow rate is so significant to LFIA performance because it determines the volume that will ultimately cross the reaction site on the membrane as well as the incubation time where the analyte forms complexes with the biorecognition element in the conjugate pad [1]. Another important component of the running buffer is the composition of the blocking buffer; a blocking buffer such as bovine serum albumin, typically at a concentration of 1-3% within the running buffer, is responsible for enhancing the running buffer's ability to control incubation time, reduce nonspecific binding, and decrease signal intensity with increasing concentration [1, 5].
2D. Other Significant Design Parameters
Aside from consideration for the antibodies and the materials for each pad, there are many other significant parameters associated with the assembly and performance of the assay that require optimization to enhance its functionality and must be kept consistent between test strips to ensure that the results are reproducible and the device's sensitivity is refined [5, 8, 17]. These include the distance between the test and control lines on the membrane, the dimensions of the strip and arrangement of the pads, and the temperature at which the assay cartridge is incubated to facilitate fluid flow; the biological components being accurately dispensed, assembling the strip components properly, and proper drying and blocking of the device being performed are also important parameters [5, 8, 11, 12, 14, 17]. Overall, it is important to keep all of these parameters, the pad materials, assay buffer compositions, the sample volume, and the amount of antibodies on the test line, control line, and conjugate pad consistent between assays; each parameter must be optimized one at a time to enhance the LFIA's overall performance are necessary for ensuring both precision and reproducibility of the results [5, 14, 20, 26]. Some assays reported in the literature do not provide all of these parameters for the final assay or explain the standard parameters utilized for the assays during the earlier stages of the optimization process [5, 6, 11, 18, 27]. The cost of materials and chemicals required for the components, assembly, and performance of the assay are also an important consideration [1, 2, 5, 7-9, 11, 14, 17].
3. Advantages and Disadvantages of LFIA Technology
As a well-established and optimized PoC diagnostic approach, there are many advantages associated with LFIA technology. Like many other PoC diagnostic technologies, LFIA testing results can be obtained rapidly after the test's initiation, all without sacrificing the test's reliability or accuracy [1, 8, 14]. This can ensure that diseases will more often be diagnosed at an earlier stage, which will speed up the time to receive proper treatment and increase the likelihood of recovery or survival [1, 3, 8]. Samples do not need to be transferred to a laboratory with large amount of additional time being needed for a skilled technician to transmit and collect the results [1, 14]. Moreover, PoC testing can be self-administered, not necessarily having to be administered by a medical professional, which provides patients with the ability to self-monitor and manage their conditions themselves, however, this requires that information to be provided to help patients understand device use and maintenance where necessary, as well as where they can find additional support for their conditions [1, 23]. A common example of a PoC LFIA that can be self-administered is a pregnancy test [1, 2, 9, 16]. Self-administered LFIA testing also reduces the need to visit the doctor, saving time and money in addition to allowing medical professionals to accommodate patients in more critical conditions in-person [1, 23]. LFIA materials, assembly, and readers are also low-cost, allowing more affordable worldwide healthcare in low and middle-income countries with limited resources, where trained personnel, expensive equipment and instrumentation, and well-equipped diagnostic facilities are far less accessible [1, 2, 5, 7, 11, 14, 18]. Public health in the developing world will therefore significantly improve with the introduction of more accessible, affordable diagnostic testing that provides rapid, accurate results; in PoC testing that is portable, does not require refrigeration for shipping and storage or complex pre-processing for the biological samples, and is highly sensitive and specific in detecting the target analyte(s) are especially viable for low resource-settings [2, 7, 8, 14, 18]. The shelf-life of this technology tends to be 12-24 months without refrigeration, though it can vary between lateral flow assays depending on their design, constituents, and environmental conditions such as temperature and humidity [12, 20]. LFIA testing is overall highly versatile and can be utilized for the detection and diagnosis of a wide variety of infectious and non-communicable diseases, requiring only a small amount of sample [2, 5, 8, 17]. LFIAs have also been utilized for other applications such as food and environmental safety as well as veterinary medicine [19, 21].
There are also many disadvantages and limitations associated with standard LFIA technology despite the fact that as a diagnostic platform, it has been demonstrated to be simple, low-cost, and highly versatile [6, 8, 9]. There is a current need to improve the sensitivity of these assays, making optimization protocols significant for establishing LFIA devices with high sensitivity and specificity [5-8, 14, 16, 17, 23]. There are concerns about errors and interferences because traditional PoC devices like LFIAs tend to be single-use test cartridges, which is why controlling the parameters of the device and batch-to-batch reproducibility are important, non-trivial factors in the manufacturing and performance of a LFIA [1, 14]. While porous nitrocellulose is widely accepted for use as a membrane material for LFIAs due to its capabilities for controlling capillary flow and protein adsorption, this material and others typically used for LFIA such as polyesters and rayon are limited by their non-specific binding properties, sensitivity to humidity and brittleness [1, 8, 12]. Nitrocellulose in particular possesses a highly flammable matrix [12]. Pre-processing of biological samples might also be necessary to improve sample compatibility with the test in the case that there are interferents that must be removed or when tissue samples are being tested rather than fluids such as blood, plasma, or urine; the need for post-processing and the specific type of sample being tested [tissue biopsy vs blood or urine) is a major determinant of whether a LFIA test can be made available as a consumer product that can be self-administered, such as how a diabetes test detects glucose levels within a blood sample [8, 16, 20, 23, 25]. Innovation is therefore needed to refine the sensitivity and reproducibility of LFIA testing in order to obtain more reliable, accurate results that confirm a disease's presence while also optimizing development costs [1, 5, 6, 8, 9, 12, 14, 17, 23].
4. Emerging Trends and Innovation associated with LFIA Technology
Besides optimization protocols, there have been many recent innovations and trends related to improving traditional LFIA technologies' performances, which include advances in materials and enabling technologies for manufacturing, LFIA design approaches, and detection strategies [1, 3, 5 8, 9, 12, 14, 15, 23, 24]. A significant trend that has been utilized in multiple studies is the strategy known as multiplexing whereby multiple analytes can be detected on a single LFIA to ensure more accurate and efficient detection of a specific disease while also allowing for the particle disease to be subtyped. Additionally, the benefits of using a multiplex strategy for high-throughput PoC testing can reduce costs and is especially viable in cases with limited sample availability and where advanced medical decision-making is necessary [1, 7-9, 23]. Several studies have explored the use of multiplexing in the design of more sophisticated LFIAs, with promising results [9, 11, 18]. Multiplexing can be performed by using an array of strips testing for the presence of different analytes, placing multiple, spatially separated test lines for the different analytes on the same strip, utilizing different detection labels with distant signals, and using a broad-selective recognition element that can bind to a class of several compounds. A combination of strategies is also a common approach. Only a maximum of six lines can be applied to a multiplexed strip (including the control) without elongating the strip, which means that only a maximum of five biomarkers can be detected; the problem with strip elongation is that a larger amount of sample and time for the assay to be completed will be required [9]. Utilizing dot-shaped detection sites instead is a feasible alternative if more than five detection sites are necessary for the assay [9].
Microfluidic systems can be used to generate powerful biological assays for use in developing countries as well; while they may eventually take the place of LFIAs as the most successful and utilized PoC diagnostic technology in the future, this emerging technology can also complement LFIAs [1, 3, 8, 12, 16, 23]. For example, inaccurate sample delivery to the strip and differences in sample and conjugate movement to the assay's reaction matrix can lead to variation in LFIA results; integration of fluidic elements such as molded capillary holder in Saliva Diagnostic Systems' HemaStrip can potentially overcome these mechanical limitations intrinsic to LFIA systems [20]. Another major trend is the use of smartphone-based PoC technologies, as smartphones are widely available technology with cameras that can be utilized as colorimetric and fluorescent readers [1, 3, 7-9, 11, 23, 27, 28]. A large number of cell phone users in developing countries, making the smartphone-based readers for LFIA applications especially viable for use in low-resource countries [3, 8, 16, 23, 28]. Use of newer optical methods are also able to increase the sensitivity of these assays by reducing background, enhancing the signal, or a combination of the two. One example is Forster resonance energy transfer (FRET), which can facilitate a proximity-dependent reaction by non-radiatively transferring energy from a fluorescently-excited donor molecule to an acceptor molecule within the donor's vicinity. No signal arises until the analyte brings the detection and capture reagents into proximity by separately binding to them; therefore, since only the combination of these two reagents can generate a signal, the signal generated by a LFA utilizing FRET will be amplified compared to a conventional LFIA [1, 19]. LFIA technology and PoC diagnostics as a whole are definitely disease-related fields with a significant amount of potential and active ongoing research [1, 3-5, 9, 10, 12, 23].
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