Sensitive biomolecule detection in lateral flow assay with a portable temperature–humidity control device
Introduction
With the rapidly increasing incidence of infectious diseases (e.g., Ebola, dengue, malaria, human immunodeficiency virus (HIV) and influenza) owing to globalization, limited access to medical services in developing countries has become a major challenge (Laursen, 2012, McNerney and Daley, 2011). To address this issue effectively, a robust system is required to bring accurate diagnostic assays to the point of care (POC). This could greatly simplify the existing laboratory-based assays (i.e., quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA)) as well as reducing the cost and time demands they bring about, especially in resource-limited settings, where most diseases exist (Burke and Gorodetsky, 2012, Han et al., 2014, Sackmann et al., 2014). Today, the use of a paper-based platform represents a promise of a simple, portable, cost-effective, and user-friendly POC diagnostic tool, which holds a great potential as an alternative to the conventional laboratory techniques (Choi et al., 2015, Song et al., 2012). Recent studies have focused on the use of lateral flow assay (LFA) for accurate POC diagnostics (Blažková et al., 2009, Hu et al., 2013, Wang et al., 2013). These assays normally involve hybridization of a single stranded-target analyte (RNA or DNA) with a complementary probe to form a double-stranded nucleic acid, or binding between antigen (Ag) and antibody (Ab) to form an Ag-Ab complex, which produces a colorimetric, fluorescent or chemiluminescent signal (Cate et al., 2014, Martinez, 2011). However, to date, the main limitations of LFA are the difficulties in quantification and lack of sensitivity (Feng et al., 2015, Hu et al., 2014).
Several efforts have been made to address these limitations. To achieve quantification, researchers have developed a variety of handheld or smartphone-based readers to quantify the LFA results (Mudanyali et al., 2012). As for the sensitivity improvement, various techniques have been developed through probe-based signal enhancement (Hu et al., 2013), enzyme-based signal enhancement (He et al., 2011), thermal contrast (Qin et al., 2012), or fluidic control (Parolo et al., 2013, Rivas et al., 2014). However, the sensitivity of LFA might be significantly affected by the environmental conditions, which have been overlooked in most studies. Accumulating evidence has shown that environmental factors (e.g., temperature and relative humidity (RH)) may have a significant influence on biomolecular reactions (Barry and DeMille, 2012, De Roy et al., 2013, Wu et al., 2014), including nucleic acid hybridization (Zhang et al., 2012) and Ag–Ab interaction (Reverberi and Reverberi, 2007). Further, RH may also influence the assay readout by affecting the fluid wicking rate in the paper, which may in turn affect the sensitivity of paper-based assays (Giokas et al., 2014, Lutz et al., 2013, Renault et al., 2013, Rivas et al., 2014). As LFAs are intended for field application, they may be affected by user’s environmental conditions (e.g., extremely hot or cold, and dry or wet environments) more than in a typical controlled laboratory. For instance, the temperature and RH of well-known dengue endemic area, Malaysia, is in the range of 25–37 °C and 70–90% RH, respectively, which may not produce an optimum assay outcome. Therefore, effective monitoring and control of environmental factors plays an important role in maintaining optimum conditions for biomolecular reactions, and in turn enhancing the analytical sensitivity of the current LFA. Several studies have investigated the stability of LFA in different environmental conditions (Chien et al., 2006, Johnson et al., 2005). However, the optimum environmental requirement for sensitivity enhancement in LFA for nucleic acid or antigen/antibody detection has not been explored yet.
The present study reports the use of a portable temperature–humidity control device to provide an optimum environmental requirement for sensitivity improvement in LFAs, followed by quantification of multiple types of targets (DNA or protein) by using a smartphone. Interestingly, in DNA detection, temperatures between 55–60 °C (representing the annealing temperature in PCR) provides the maximal DNA hybridization signal, without significantly affecting the shape of the paper (e.g., deformation), whereas an RH beyond 60% could effectively facilitate the fluid to completely wick through the paper to produce the desired signal. With optimum experimental conditions (55 °C, >60% RH) our lateral flow test strip was able to improve the sensitivity of almost 10-fold compared to that achieved at ambient conditions (25 °C, 60% RH), using dengue viral DNA and HIV DNA as model analytes. We have also successfully shown the optimum Ab–Ag interaction at 37–40 °C by using this simple test strip. Given that precise temperature and humidity control is technically challenging outside the laboratory, we developed a portable temperature–humidity control device to achieve optimum LFA performance in a POC setting (Fig. 1). We envision that in the future, the integration of a fully integrated paper-based sample-to-answer biosensor into this portable device offers great potential for sensitive detection of various target analytes in resource-poor settings.
Section snippets
Preparation of gold nanoparticles (AuNP) and AuNP-DP (detector probes) conjugates
Gold nanoparticle (AuNP) with diameter of 13±3 nm were prepared according to the previously published protocol (Hu et al., 2013). In a 250 mL round-bottom flask, 4.5 mL of 1% tri-sodium citrate and 1.2 mL of 0.825% chloroauric acid was added to 100 mL-boiled ultrapure water. The solution turned from yellow to purple and finally turned wine-red in 2 min. The solution was used to prepare AuNP-DP conjugates. Both AuNP and AuNP-DP conjugates were characterized by UV/Vis spectrophotometry and TEM (TEM,
The working principle of lateral flow assay in a temperature–humidity-controlled manner
LFAs are commonly performed based on nucleic acid hybridization or Ag–Ab reactions. Both test strips are made up of a nitrocellulose membrane containing a test zone and a control zone, a sample pad, a conjugate pad deposited with AuNP-DP, an absorbent pad to facilitate the fluid flow and a backing pad as a support of the strip. In nucleic acid detection, the target DNAs are allowed to bind to the AuNP-DP, forming AuNP-DP-target DNA complexes, which in turn interact with the capture probe at the
Conclusion
In short, with the simple temperature–humidity control device, we successfully proved that temperatures of 55–65 °C and 37–40 °C, with a humidity of 70–90% give an optimum signal for nucleic acid and antigen detection in LFA respectively. This compact, portable and cost-effective temperature–humidity control model, coupled with the test strip, offers rapid, specific and sensitive diagnosis of infectious diseases in POC settings. We expect that our prototype will raise concerns about the
Acknowledgement
This work was financially supported by the Major International Joint Research Program of China (11120101002), the International Science & Technology Cooperation Program of China (2013DFG02930), and the Ministry of Higher Education (MOHE), Government of Malaysia under the high impact research (UM.C/HIR/MOHE/ENG/44). FX was partially supported by the China Young 1000-Talent Program and Program for New Century Excellent Talents in University (NCET-12-0437). The work was performed at Bioinspired
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