Elsevier

Biosensors and Bioelectronics

Volume 96, 15 October 2017, Pages 213-219
Biosensors and Bioelectronics

A Point-of-Need infrared mediated PCR platform with compatible lateral flow strip for HPV detection

https://doi.org/10.1016/j.bios.2017.04.047Get rights and content

Highlights

  • Conducting oil and carbon nanotube was used as medium to transfer heat from IR-LED to PCR samples.

  • This platform offers accurate and flexible control of temperature through the integration of PID algorithms.

  • The performance of this portable and inexpensive platform is a match to the conventional bench-top thermo cycler.

  • Lateral flow stripe was used to detect the PCR products.

Abstract

With the increasing need of monitoring the epidemiology of serious infectious diseases, food hygiene, food additives and pesticide residues, it is urgent to develop portable, easy-to-use, inexpensive and rapid molecular diagnostic tools. Herein, we demonstrate a prototype of IR mediated Conducting Oil and CarbOn Nanotube circUlaTing PCR (IR-COCONUT PCR) platform for nucleic acid amplification. The presented platform offers a new solution for miniaturized PCR instruments with non-contact heaters by using conducting oil and carbon nanotube as a medium in IR mediated PCR. This novel platform offers accurate and flexible control of temperature through the integration of PID (proportional–integral–derivative) algorithms to manipulate the duty cycle of the voltage signals of IR LED and a peristaltic pump. The ramping rate of the introduced platform in current study is 1.5 °C/s for heating speed and −2.0 °C/s for cooling speed. This platform fulfills 30 thermal cycles within 50 min which is a match to the conventional bench-top PCR thermo cyclers. For demonstration purpose, human papillomavirus (HPV) patient cervical swab specimens were examined. Downstream lateral flow strip (LFS) was also developed to quantity the PCR products from the IR-COCONUT PCR device within 25 min. This PCR platform together with the compatible LFS shows great potential for in-field and Point-of-Need (PoN) testing of genetic or contagious diseases.

Introduction

Point-of-Need (PoN) testing, a new concept which has been proposed to enrich Point-of-Care (PoC) testing, is defined as any diagnostic test that can be performed out of reference laboratories, such as home, patient's bed, ICUs, physician's office or production line, etc. (Benjamin and Sébastien, 2016). PoC testing relates to human testing whereas PoN testing refers to a much larger scope, including human testing (Chuang et al., 2016; Lee et al., 2015; Wang et al., 2012), veterinary testing (Tarasov et al., 2016), industrial testing (Wang et al., 2017), argo-food testing (Raeisossadati et al., 2016), environmental testing (Bidmanova et al., 2016; Gomes and Sales, 2015), forensics (Soejima and Koda, 2014) and so on. Since the first applications of PoC testing in 1990s, microfluidic technology has been used to solve technical problems and it has become part of the diagnostics revolution in the healthcare industry. The emerging field of microfluidic technology in combination with Micro-Electro-Mechanical Systems (MEMS) can provide exciting solutions to PoN testing. Bio-analytical systems based on microfluidics and MEMS can allow for miniaturization and integration of processes onto a chip with benefits including test speed, cost, portability and automation.

With the increasing need of monitoring the epidemiology of serious infectious diseases, food hygiene, food additives and pesticide residues, particularly in developing countries with poor infrastructure or on-site detection with the need of rapid results, it is urgent to develop portable, easy-to-use, inexpensive and rapid molecular diagnostic tools. In recent advances, lateral flow assays (LFAs) (Ahrberg et al., 2016, Choi et al., 2016, Raston et al., 2016, Rodriguez et al., 2016) have been used to achieve simple and cost-effective nucleic acid testing. LFAs are capable of producing semi-quantitative results by fluorescence signals or visible color formation in less than 30 min. However, the constituent of biological samples (e.g., blood, urine and saliva) are generally complex and contain low amounts of target nucleic acids. Prior to lateral flow detection, the sample pretreatment, off-chip extraction/solid-phase extraction and amplification processes (polymerase chain reaction (PCR)) are normally required.

Miniaturized PCR instruments (Ahrberg et al., 2016, Chung et al., 2010, Liu et al., 2004, Park et al., 2016) utilize both MEMS and microfluidic technology to fulfill the conventional PCR functions onto a portable device that can be used in any condition and capable of rapid target gene identification. Many miniaturized PCR devices have exhibited superior performance than bench-top PCR thermo cyclers in the aspects of cost, ramping speed, portability, integration level, efficiency, etc. Based on the design of sample chambers, miniaturized PCR instruments can be categorized into static chamber PCR (Sposito et al., 2016, Zhu et al., 2014) and continuous flow PCR (Chung et al., 2010). The static chamber PCR resemble the bench-top PCR thermo cyclers as the sample is kept in a small chamber and undergoes a continuous temperature cycle. The sample in continuous flow PCR travels repeatedly through three temperature zones for amplification purpose. The design of static chamber PCR could be more impact than the continuous flow PCR and the number of cycles is easier to alter based on demands. However, the temperature ramping rate of static chamber PCR is lower than the continuous flow PCR. Over the years, various on-chip heaters have been demonstrated to improve the speed of temperature cycling. In general, the heaters have two categories, namely contact type (thin films, metal heating blocks and Peltier units) and non-contact type (IR, microwave and laser heater units). The ramping speed of micro-fabricated thin film can achieve 175 °C/s for heating speed and −125 °C/s for cooling speed and fulfill 40 thermal cycles within 6 min (Neuzil et al., 2006). But the contact heaters have the demerit of complexity and cost of fabrication. On the other hand, the compact heater design which reduces the thermal mass of the whole chip is essential to achieve fast temperature cycling speed. The non-contact heaters are more favorable for simple design and flexible system integration. As the heaters do not have to contact the sample chamber, it offers more opportunities to utilize multiple chip designs upon the same platform. Several researchers have successfully integrated IR-mediated (Hühmer and Landers, 2000, Hagan et al., 2011, Legendre et al., 2006) or laser assisted heater units (Slyadnev et al., 2001) to directly heat the PCR sample and achieved amplicon detection. The previous IR-mediated PCR utilizes a high-power tungsten lamp and convex lens to heat the sample directly. This method suffers a great heat loss due to the large thermo mass and has a limitation to conduct multi-chamber PCR at the same time.

In the present study, we developed a prototype of IR mediated Conducting Oil and CarbOn Nanotube circUlaTing PCR (IR-COCONUT PCR) platform for nucleic acid amplification. This platform can fulfill both thermal cycling like three stage/two stage PCR and isothermal amplification like Loop-Mediated Isothermal Amplification (LAMP), Strand Displacement Amplification (SDA), Helicase Dependent Amplification (HDA), etc. For demonstration purpose, human papillomavirus (HPV) was applied as a model analyte to examine the three-stage temperature cycle performance of this IR-COCONUT PCR. The PCR products were detected by lateral flow strip with less than 25 min.

Section snippets

Microfluidic chip design and fabrication

The structure of PCR microfluidic chip is illustrated in Fig. 1A. This chip was constructed using a five layer stack consisting of an upper cover layer, a PCR chamber layer, a copper layer, a conducting oil layer and a lower cover layer. All the four layers apart from the copper layer were made of PMMA with 1 mm thickness. In the PCR chamber layer, three identical oval shaped channels (20 μL) were fabricated (Fig. 1B) to expel dead zone air when introducing samples to the channels. The middle

Comparison between IR-COCONUT PCR and conventional bench-top PCR

The heating and cooling speed and accuracy of temperature is influenced by several parameters. Firstly, the LED light power, carbon nanotube concentration, maximum flow rate of the peristaltic pump need to be decided. For instance, the LED power should be high enough to heat up the conducting oil to more than 95 °C in a short time and it should be low enough to save electricity power. The most important optimization of the temperature is to find the proper PID parameters (proportional term,

Conclusions

We have developed a novel PCR platform named IR-COCONUT PCR. This novel platform offers accurate and flexible control of temperature through the integration of PID algorithms to manipulate the duty cycle of the voltage signals of IR LED and a peristaltic pump. The ramping rate of the introduced platform in current study is 1.5 °C/s for heating speed and −2.0 °C/s for cooling speed. This platform fulfills 30 thermal cycles within 50 min which is a match to the conventional bench-top PCR thermo

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81301293) and China Major New Drugs Development Program (2014ZX09507008).

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