Mini-thermal platform integrated with microfluidic device with on-site detection for real-time DNA amplification
Abstract
The recent cases of COVID-19 have brought the prospect of and requirement for point-of-care diagnostic devices into the limelight. Despite all the advances in point-of-care devices, there is still a huge requirement for a rapid, accurate, easy-to-use, low-cost, field-deployable and miniaturized PCR assay device to amplify and detect genetic material. This work aims to develop an Internet-of-Things automated, integrated, miniaturized and cost-effective microfluidic continuous flow-based PCR device capable of on-site detection. As a proof of application, the 594-bp GAPDH gene was successfully amplified and detected on a single system. The presented mini thermal platform with an integrated microfluidic device has the potential to be used for the detection of several infectious diseases.
Method summary
This study describes an automated, integrated and miniaturized microfluidic continuous flow-based PCR device accomplished for on-site detection of infectious and noninfectious genomic templates. The microfluidic device was fabricated using a dry film photoresist substrate patterned on a glass slide using a direct laser writer. The detection unit works on the principle of photometric aspect. Here, a 20-μl PCR mixture along with SYBR® Green-I dye was infused into the microfluidic device via an automatic syringe pump with an optimized flow rate and time frame of 5 μl/min and ∼18 min, respectively.
In recent times, there has been tremendous interest in the development of miniaturized bioanalytical instruments for a variety of point-of-care (POC) diagnostic applications. Such miniaturized devices have attracted a lot of interest because they offer several benefits, including minimal volumes of reagents (∼20 μl), short reaction times (∼18 min), low power consumption (12V), rapid analysis (photometric-based) and cost–effectiveness (∼$47) [1,2]. One of the main constituents of such devices is a microfluidic device or lab-on-a-chip to integrate different functions of a biological test – such as sample preconditioning, chemical reaction, sample separation and detection – into a single channel. In this context, one of the crucial biological assays, PCR-based nucleic acid amplification, can also be realized for POC diagnostics by leveraging the unique facets of microfluidics or lab-on-a-chip devices [3–5]. Usually, nucleic acids can be amplified in two well-established techniques: isothermal-based and thermal gradient-based approaches. Isothermal amplification is an approach used for the amplification of RNA and DNA samples that are broadly used in biological tests. It permits simplification in temperature control because the temperature remains constant. Moreover, its mechanism uses a reasonably low temperature for the amplification of nucleic acids in a short period of time. The isothermal approach can be classified into several types: loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification, rolling circle amplification, multiple displacement amplification and nucleic acid amplification technologies [6–9]. Table 1 shows the differences between isothermal and PCR techniques.
| Criterion | Isothermal | PCR |
|---|---|---|
| Temperature range | <60°C | >90°C |
| Number of primers | Four to six specifically designed primers | Two primers |
| Reaction time | Up to 45 min | Up to 1–2 h |
| Visual detection | Possible | Impossible |
| DNA output | 10–20 μg yield | Up to 0.2 μg yield |
| Economy and ease of use | Economical, easy to operate | Expensive, requires skilled operator |
| Knowledge of the method and clinical evaluation | Limited, with ongoing evaluation | Well-known and clinically proven |
| Sample inhibitor sensitivity | Insensitive | Sensitive |
PCR is an essential technique for genetic analysis, usually carried out by thermocycler; it suffers from long assay times and typically takes 1–4 h, because of slow transition rates in thermal parameters [10,11]. To overcome these problems, an ideal miniaturized PCR device should allow for rapid thermal cycling at a minimum reaction volume. Typically, during the PCR process, in the presence of an enzyme, primers and dNTPs, the DNA molecules are amplified using three thermal zones: denaturation (94°C), annealing (55–60°C) and extension (72°C). The COVID-19 pandemic has spurred the prospect of and need for the development of miniaturized PCR devices that can be used for rapid and easy detection of pathogens and viruses [12]. The proposed device has the potential to be used to detect both infectious and noninfectious pathogens. In most miniaturized PCR devices, the reaction sample is introduced into a microchamber, and the DNA amplification is performed by thermal cycling. However, chamber-based heating and cooling rates are relatively slow in the process, and the chances of sample evaporation are quite high [13]. Conversely, a continuous-flow-based PCR device involves transferring a reaction sample through a capillary column in a microfluidic channel and having three thermal zones maintained at the desired temperature, thus effectively addressing the problem of slow heating rates. The features of a continuous flow-based PCR apparatus include rapid and consistent temperature control as well as a reduced reaction time [14].
In many cases, microfluidic technology has led to the increasing popularity of POC testing platforms for biological and clinical applications [15,16]. Microfluidics uses a fluid in a micro- or nano-fabricated capillary which controls and manipulates the fluid, even down to the nanoliter scale, flowing within the microchannels. Microfluidics also relates to the design and development of micro-devices that move or analyze the small volume of a reaction sample [17]. Without the need for a competent operator, microfluidic devices can perform tests on microscale volumes, including on complex fluids, with high efficiency and speed [18]. In addition, the fabrication of such microfluidic PCR devices brings several benefits, including facilitating the use of low sample volumes, allowing for high-throughput and rapid thermocycling, and being economical. Because of the high surface-area-to-volume ratio associated with microstructures, this technique has drastically boosted heat transmission rates [19–21].
Several materials have been used to fabricate microfluidic devices, such as silicon, polymers, glass, polyimide and quartz [22–25]. One of the polymer materials which is most commonly used in biological assays is polydimethylsiloxane (PDMS). PDMS has several benefits, including biocompatibility and cost–effectiveness, because a simple replica molding technique is used that allows mass production of a microfluidic device. Thus the microscopic observation of the sample inside the microchannel is possible. However, polymers have a low thermal conductivity, which may slow down the heating and cooling rates. The general pipeline for the design and development of microfluidic devices includes selection of material; optimization of the design; and fabrication of the device, using several techniques (e.g., photolithography, soft lithography, etching, lithography, CO2 laser ablation, Computerized numerical control (CNC) milling, 3D printing and UV direct laser writers [UV-DLWs]) [26–29]. Table 2 illustrates several materials utilized to develop microfluidic devices.
| Materials | Melting point | Thermal conductivity (W/mK) | Advantages | Disadvantages | Ref. |
|---|---|---|---|---|---|
| Polydimethylsiloxane | >200°C | 2.73 | • Optical transparency • Low cost • Simple fabrication process • Conformal contact achievable on nonplanar surfaces • Permeable to a variety of liquids and vapors • Excellent thermal stability | • Wide and shallow microchannels easily collapse during bonding • Tends to shrink by 1% upon curing | [30] |
| Polymethylmethacrylate | 150°C | 0.17–0.19 | • Excellent transparency • High mechanical strength and hardness • High rigidity • Good thermal stability • Low water adsorption | • Brittle • Low impact resistance • Low chemical resistance • Possibility of stress problems • Requires additional instrument to fabricate | [31] |
| Polycarbonate | 265°C | 0.19–0.22 | • Extremely high impact strength • Superior clarity | • Scratches easily • High cost | [32] |
| Polytetrafluorethylene | 327°C | 0.25 | • High-temperature tolerance • Exceptional lubricity • Caustic fluid inertia | • Very soft | [33] |
| Polypropylene | 160°C | 0.1–0.22 | • Inexpensive • Semicrystalline nature • Low coefficient of friction • Good chemical resistance | • High thermal expansion coefficient • High flammability | [34] |
| Polyimide | 250°C | 0.10–0.35 | • Superior temperature adaptability • High chemical resistance • High mechanical performance | • Expensive fabrication | [35] |
| Polyethylene terephthalate | 260°C | 0.15–0.4 | • Higher strength and stiffness • Lightweight • Excellent electrical insulating properties | • Very susceptible to heat degradation | [36] |
| Glass | 1200°C | 0.76 | • Cheaper • Good protection power • Outstanding transparency • Great heat resistance | • Fragile • More weight | [37] |
| Silicone | 350°C | 0.2 | • Excellent thermal stability • Good flexibility • Low chemical reactivity • High efficiency | • Brittle • Expensive for a single substrate | [38] |
| Paper (cellulose) | 220°C | 0.05 | • Very cheap • Easy to process materials • Fluid flow is automatic • Biodegradable | • Low resolution • Limited to simple designs | [39] |
Evidently, a thermal management system is extremely important and widely used in heating and cooling applications [28]. Apart from this, several other research fields also require thermal management systems (e.g., biomedical, nanomaterial synthesis and biochemical domains). During DNA amplification using the PCR technique, precise regulation of temperature is very important for better sample yield. There is a considerable need for a portable thermal management system with accurate, stable, easy-to-use and low-cost microdevices that can be used for biological diagnostic applications in a microfluidic device.
Most recently, several methods and techniques have been reported by researchers and scientists for a continuous-flow PCR based on microfluidic technology, and there is an ongoing process to come up with new solutions. For instance, Cai et al. detected infections including Staphylococcus aureus and Pseudomonas aeruginosa; it took nearly 3 h from sample extraction to detection, using a microfluidic system that combined dielectrophoresis with a microfluidic multiplex PCR array [40]. Kulkarni et al. demonstrated a polymethylmethacrylate (PMMA)-based microfluidic device consisting of 30 thermal cycles fabricated using a CO2 laser engraver for amplifying the GAPDH gene with standard gel electrophoresis-based detection [41]. Ma et al. described a microfluidic droplet PCR system for amplifying particular peanut gene segments to detect foodborne diseases using fluorescence-based on-site detection [42]. Li et al. demonstrated a microfluidic device with fluidic properties, minimum resistance and double-layer-based droplet continuous-flow PCR for bacteria detection [43]. The proposed microchannel resolved the issue of the generation of bubbles and evaporation of reagents during the PCR within the capillary flow by generating nanoliter droplets. The target gene length of a periodontal pathogen was effectively quantified and determined within 11 min 16 s using fluorescence. Yang et al. described a continuous-flow PCR array microfluidic device with a multiplexing approach that can amplify more than one target gene. Within 8 min 5 s, the microfluidic device successfully amplified three target genes with an array-based method [44].
The present work describes the development of a portable, automated, battery-operated, easy-to-use and low-cost thermal management system integrated with a UV-DLW and soft lithography microfabricated continuous flow-based microfluidic device. The proposed device incorporates an on-site photometric-based detection unit using a photodiode and light-emitting diode (LED) [37]. A transimpedance amplifier circuit was developed to strengthen the signal received from the photodiode output with reduced noise. The device was integrated with an Arduino Mini Pro-based microcontroller to regulate and monitor the thermal management system. A self-designed driver circuit was used for the process to switch action across the cartridge heater. Further, Internet of Things (IoT) and Bluetooth modules were mounted onto a single printed circuit board (PCB) platform and used for live data logging, accessed on a smartphone. As a proof of concept, the developed miniaturized platform was used to amplify the 594-bp rat GAPDH gene effectively. The amplified output was detected on a single integrated mini-platform. Herein, the novelty of the proposed work includes reduced reaction time, excellent device sensitivity and a fully integrated and automated device with on-site detection unit. Table 3 summarizes the most recent trends in the development of continuous-flow based microfluidic devices compared with the salient features of the proposed work.
| Parameter | Baudrey et al. [45] | Kulkarni et al. [46] | Xu et al. [47] | Qin et al. [48] | Current work |
|---|---|---|---|---|---|
| Fabrication and material | Photolithography/PCB | CO2 laser/PMMA | CNC milling/soft lithography | Hot embossing/COC | UV-DLW/soft lithography |
| Channel dimension (L × W × H), mm | 76 × 52 × 1 | 30 × 0.5 × 0.5 | 20 × 0.24 × 0.25 | 46 × 30.9 × 0.4 | 30 × 0.42 × 0.035 |
| Reaction volume | 30 μl | 20 μl | 30 μl | 20 μl | 20 μl |
| Thermal loops | 35 | 30 | 30 | 40 | 30 |
| Reaction time | 45 min | 32 min | 38 min | 20 min | 18:41 min |
| Detection method | Gel electrophoresis | Electrochemical-based | Gel electrophoresis | Fluorescence-based | Photometric-based |
Materials & methods
Chemicals & apparatus
DreamTaq Green PCR 2× Master mix and 1 Kb Plus Invitrogen DNA ladder were purchased from Thermo Fisher Scientific (Bengaluru, India). The biochemicals (Tris base buffer, acetic acid, SeaKem LE Agarose and isopropyl alcohol solvent) were purchased from SRL Pvt Ltd (Secunderabad, India). Ethidium bromide and SYBR® Green-I dye were purchased from Sigma-Aldrich (Bengaluru, India). PMMA was purchased from Industrial Polymers Corporation (Hyderabad, India), and PDMS Sylgard™ 184, Dow Corning was procured from Prabha Trade Impex Pvt. Ltd (Secunderabad, India). Table 4 shows the primer sequence of the template DNA to be amplified. The primer and template were obtained from the Department of Pharmacy, BITS-Pilani, Hyderabad Campus, India.
| Target gene | Primer sequence |
|---|---|
| Rat GAPDH – Housekeeping gene constitutively expressed, isolated from NRK-52E kidney epithelial cells, non-infectious | 5′-CAGTGCCAGCCTCGTCTCAT-3′ (forward) 3′-AGGGGCCATCCACAGTCTTC-5′ (reverse) |
A 96-well thermal cycler (Veriti™ Applied Biosystems, Waltham, MA, USA) was purchased from SR Life Science Solutions. Gel electrophoresis and gel documentation systems were purchased from BioBee Tech Solutions (Bangalore, India). A customized cartridge heater was purchased from Ragatiya Heaters (Mumbai, India). The UV-DLW tool and automated syringe pump (HO-SPLF-01) were procured from Holmarc Opto-Mechanics Pvt. Ltd (Kochi, India), Negative dry film photoresist (DFR) (1.5 mil of thickness) was procured from RistonPM 240, DuPont (DE, USA). Corning Borosil glass slide (75 mm length [L] × 50 mm height [H]) was procured from Prabha Trade Impex Pvt. Ltd (Secunderabad, India), Photodiode (S1087) was purchased from Hamamatsu Photonics (Shizuoka, Japan). Electronics components such as LED (VLWTG9900, Vishay Semiconductors) and Opamp (LM358P) were purchased from Element14 and CUTE-1MPR for Oxygen Plasma Instrument was purchased from Femto Science Inc., Hwaseong, Korea.
Design & fabrication of microfluidic device
The serpentine design of the microfluidic device was confirmed after the initial literature study involving the microchannel geometry for nucleic acid amplification with discrete microchannel parameters like structure, length, width and height [49]. Using Autodesk Fusion 360 software (Autodesk, San Francisco, CA, USA), a serpentine-based microchannel was created and stored in .dxf file format. The .dxf file was then converted to G-code using the LazyCam (BETA-release, Artsoft Inc, IL, USA) UV-DLW program, allowing for scalability. This converted file was loaded into the Mach-3 loader program, which links the UV-DLW instrument and the computer. With 30 thermal cycles, the planned serpentine microchannel has dimensions of 30 mm (L), 0.42 mm (width [W]) and 0.035 mm (H). Figure 1A shows the design of the serpentine microchannel. Figure 1B shows the DFR-based developed microchannel master on the borosil glass substrate. Figure 1C & D shows the microchannel dimensions, determined using an optical microscope (DM2000LED, Leica Biosystems, Wetzlar, Germany).
(A) Design of the serpentine microchannel. (B) Developed microchannel master on the borosil glass substrate. (C & D) Microchannel dimensions determined using an optical microscope.
Figure 2 shows the fabrication steps involved in the development of a microfluidic device using the UV-DLW instrument (to create the master) and soft lithography (to create the replicated microfluidic device) technique. A Corning borosil glass slide (75 × 50 × 1 mm) was utilized as a substrate to create a microchannel master using a negative DFR. The glass slide was cleaned with 70% ethanol and dried using oxygen gas during this process. After this, a DFR layer was placed on top of this glass slide, and both were subjected to the laminator at 110°C, ensuring no air bubbles, so that the single-layer DFR of thickness 35 μm was coated onto the glass slide for the development of the master using the UV-DLW tool. Further, the optimized parameters, like intensity, speed and focus level values, well-established by our group earlier [49], were used as a reference to acquire the micro patterns on the glass slide by exposing it to UV-DLW. The protective coating of DFR was removed when the laser writing was completed. This was created by spraying 0.80% sodium carbonate in water on a glass slide for roughly 50–60 s. Post-cleaning, the glass substrate was dried with oxygen and an optical microscope was used to determine the dimensions of the produced micropatterns. Herein, the DFR substrate was patterned on the glass slide that was used to create serpentine-based microchannels using PDMS.
Subsequently, PDMS material was used for fabricating the microfluidic device using a soft lithography technique. Micropatterned PDMS was peeled off carefully from the small container and subjected to the bonding process using a new clean glass slide. An oxygen plasma technique was used to successfully bond the microfluidic device, confirming no leakage within the glass–PDMS microchannel by passing deionized water. The microchannels were regularly cleaned with deionized water so that the microfluidic devices could be reused. It was dried in a hot-air oven at 65°C for 25–30 min while being purged with oxygen. PDMS has low thermal conductivity during the experimental conduction, with slow heating and cooling rates; however, this was overcome by using thermal plaster (Heat plaster, STARS-922, Shanghai, China) to enhance the thermal heating rate. Here, the thermal plaster was applied on the top surface of copper heating blocks, and the microfluidic device was placed on this setup to boost the heating process.
Development of miniaturized temperature controller with on-site detection unit
Figure 3 shows the block diagram of an integrated microfluidic device with thermal management and detection system. It comprises three units: portable thermal management unit, detection unit and smartphone-based data logging unit. The cartridge heater, temperature sensor, driver circuit and transimpedance amplifier circuit are all controlled by an Arduino Pro Micro-based microcontroller (Turin, Italy), which acts as the heart of the entire designed circuitry. A driver was used to control the excessive current flow across the heater (also known as overcurrent, wherein a larger than intended electric current flows through a conductor due to the load connected across the closed circuit). A custom-made cartridge manufactured with stainless steel of high grade-16 with device dimensions of 25 × 3 mm (L × diameter) was used as a heating element. A resistance temperature detector, based on a PT100 sensor, was used as a temperature sensor that changed resistance with a temperature change, acting as a feedback loop to the microcontroller. A resistance-to-digital converter breakout module MAX31865 (Maxim integrated, San Jose, CA, USA) was used to minimize the temperature signal sensing volatility. The detection unit comprises an LED and photodiode connected to the transimpedance circuit to amplify the output of the photodiode. The programming of the thermal management and detection system with IoT (ESP-8266-01) and Bluetooth (HC-05)-based data logging facility was executed in Arduino IDE open-source software.
Figure 4A shows a 3D schematic representation of the integrated miniaturized device, indicating all the electronic components used in its development. Figure 4B shows a single PCB on which all of the necessary electronic components are integrated and mounted. Herein, the thermal management system works on the proportional–integral–derivative (PID) controller that has three parameters Proportional gain (Kp), Integral gain (Ki), and Derivative gain (Kd) that are important for output performance. The output was accomplished and improved by optimized values of PID such as Kp = 190, Ki = 1 and Kd = 50 as per the requirement with very low tolerance; this was achieved using Arduino IDE library, an open-source software [50,51]. A PID controller is a control-loop mechanism employing feedback that is widely used in industrial control systems. Generally, a PID controller provides a stable, reliable and quick output [52]. Temperature sensitivity of the proposed device was ±0.5°C. Systems similar to our proposed system are used in many other domains (e.g., biomedical, biochemical, pharmaceutical, clinical and electronics). The key advantages of this proposed thermal management system are that it is robust, reliable, reusable, sensitive, low-cost and portable, with a simple user interface. Further, it can be enhanced by incorporating a smartphone-based power supply rather than connecting an adapter to power the device.
(A) 3D schematic representation of the integrated miniaturized device. (B) Electronic components mounted on the single printed circuit board.
PCB: Printed circuit board.
In earlier studies, the miniaturized thermal management system has been well-recognized and reported by our research group and has been used as a temperature controller for various applications such as nucleic acid amplification using the PCR technique and nanomaterial synthesis in a microfluidic device [53,54]. However, this current work includes an enhanced version with reduced reaction time and on-site detection technique, making it a fully automated and integrated device for POC testing applications.
Transimpedance circuit for the on-site detection unit
Figure 5A shows the transimpedance amplifier circuit, which converts the input current to a proportional output voltage in the detection unit, which was designed using an operational amplifier (LM358P) to ensure low noise, excellent repeatability and linear gain by strengthening the photodiode output. A combination Positive-Intrinsic-Negative (PIN) photodiode (S1087) and 7.6 mm of green LED with 1.75 cd (VLWTG9900) were used to detect the amplified output. The photodiode was selected such that the photocurrent changed in a linear range with irradiance, and the voltage across the resistor (30 MΩ) was measured to acquire the indicative intensity value. Here, a variable resistor was used to regulate the current being supplied to the LED to calibrate the output from the amplifier circuit in the detection unit. The value of the feedback resistor was designed, calculated and selected based on the reverse light current specification of the photodiode in order to produce the desired output from an opamp in the range of 0.1–4.9 V, which is fed to an analog pin of the Arduino that regulated and communicated with the amplifier circuit. Further, to decrease noise in the output, resistors were selected to establish a bias at the non-inverting terminal. The input voltage fed to the LED was varied by the variable resistor (10 KΩ) to change the intensity of the light emitted by it. The input voltage of the LED is analyzed by the output voltage from the amplification circuit.
(A) Schematic of the transimpedance amplifier circuit. (B) Schematic representation of the detection unit. (C) Amplifier output voltage (Vout) versus light-emitting diode input voltage (Vin).
The amplifier output showed good repeatability and linearity with the input voltage of the LED. Figure 5B shows a schematic representation of the detection unit wherein the PMMA microchamber of dimension 50 (L) × 50 (W) × 6 mm (φ) with a thickness of 2 mm was positioned in between the LED and photodiode. The amplified sample was collected automatically due to pressure-driven flow in the microreservoir for detection. Figure 5C shows the relationship between amplifier output voltage (Vout) and LED input voltage (Vin), with a correlation coefficient R2 = 09983. This signifies good precision and excellent repeatability of the obtained data to calculate the absorbance of the sample to be measured. This experiment was repeated for two or three iterations and each time showed the same linearity of the proposed detection unit. The collected data to determine the absorbance of an amplified sample had good accuracy and repeatability. Furthermore, by adjusting the input voltage of the amplifier, the output voltage was studied in relation to the incident light from the LED.
Cost estimation analysis
The cost estimation analysis of the proposed device is presented in Table 5. The proposed system was also compared with the existing commercially available thermocycler using parameters including size, cost, precision, portability and power consumption. In all these aspects, the proposed device was preferable and more affordable. Furthermore, it has additional features that include a PID controller, IoT for real-time data logging and an on-site photometric-based detection unit.
| Sl.No | Component | Price (US$) |
|---|---|---|
| 1 | Arduino board | 2.84 |
| 2 | MAX31856 | 7.89 |
| 3 | PT100 sensor | 1.56 |
| 4 | s8050 | 0.21 |
| 5 | IRFZ44N | 0.88 |
| 6 | Cartridge heater | 4.76 |
| 7 | Resistors and connectors | 0.14 |
| 8 | Opamp (LM358P) | 9.52 |
| 9 | Photodiode (s1087) and green LED | 8.74 |
| 10 | PDMS (1 kg) | 2.21 |
| 11 | Borosil glass slide (75 × 50 × 1 mm3) | 2.09 |
| 12 | PMMA (1/2/4 mm) | 1.84 |
| 13 | DreamTaq PCR Master Mix (2×) | 2.45 |
| 14 | Dry film photoresist | 2.70 |
| Total estimation | 47.83 | |
Results & discussion
Integrated temperature controller & detection unit
Figure 6 shows the fully integrated and miniaturized continuous flow-based microfluidic PCR for nucleic acid amplification and detection on a single platform. A copper heating block of dimension 80 × 15 × 10 mm was used as a heating platform. A layer of thermal paste was gently applied on top of the copper block surface before a microfluidic device was placed on it. The thermal conductivity of the glass- and PDMS-based microchannel was improved because of its contact with the copper block along with thermal paste; the heat exchange was more efficient, resulting in a faster heating process. The battery unit consists of three Li-Ion 18560 batteries (3.7 V/3500 mAh) coupled in series to achieve the summed-up voltage, further connected to the battery charger (TP4056) and DC boost converter (XL6009) module to generate the required 12 V by the device. When the batteries are fully charged, the device can function for more than 3–3.5 h without any power cut or fluctuation. A 4-mm transparent PMMA sheet was used for the device packaging, which was covered with a polyvinyl chloride adhesive pure black sheet on top of the PMMA sheet. The proposed device dimensions were 15 (L) × 10 (W) × 5 cm (H). An external syringe pump was used for infusing the sample onto the microfluidic device, but in the future this can be overcome with an integrated micropump unit, showing the path for a fully miniaturized module.
Data transmission & smartphone-based storage
Figure 7A shows the real-time data-logging facility connected to the smartphone using the IoT (ESP8266) module. The temperature values of the thermal management unit (denaturation [94°C] and annealing [60°C]) and the amplified absorbance value of the detection unit were directly accessed and stored on the IoT cloud server. The third temperature value, corresponding to the extension zone, was obtained by a balancing strategy and was checked using a thermal imaging camera. Figure 7B shows a uniform temperature distribution and good thermal gradient across the microfluidic device. The temperature gradient defines the change in the physical quantity that occurs with direction and temperature at a specific rate across a particular zone; by measuring the intensity change over the distance of the microfluidic device, from left to right, this thermal picture was employed for calculating the temperature gradient between denaturation (94°C) and annealing (60°C) zones. ImageJ (NIH, MD, USA) was used to determine the intensity of specific zones. This study was repeated for two or three iterations to understand the temperature gradient uniformity with different thermal images. However, it requires some initial time to reach the desired temperature based on the ramping rate and heating block dimensions. Figure 7C shows the temperature versus time plot for both the cartridge heaters at an applied voltage of 12 V. Time dependency of the temperature can be noticed. The heater exhibited a decent ramping rate over the applied power: the required warm-up time was approximately 6 min for the entire copper block to influence the higher degree of temperature for the thermal cycling process, after which it remained stable throughout the process. Here, the warm-up time is primarily dependent on the length of the copper heating blocks. Once it reaches the desired temperature, the device performance is stable and saturated for an infinite time with varying temperature accuracy of ±0.5°C.
(A) IoT-based data logging. (B) Gradient of temperature between denaturation and annealing thermal zones (analyzed using ImageJ). (C) Temperature versus time plot for both the cartridge heaters at denaturation (95°C) and annealing (60°C), respectively. IoT: Internet of Things.
Microfluidic device temperature profile
An infrared camera (PTi120, Fluke corporation, Everett, WA, USA) was used to capture the surface temperature profile of the microfluidic device. Figure 8 shows the thermal images of the microfluidic devices depicting three thermal zones of the PCR: denaturation (94°C), annealing (60°C) and extension (72°C). The extension zone was obtained by equilibrium conditions from the two set temperatures; that is, no active heating was carried out underneath the microchannel, and the third temperature is achieved by equilibrium. In Figure 8, a larger density of temperature profile may be easily differentiated and noticed. The site point confirms the real temperature of that particular temperature zone in the thermal image. As a point of tolerance for nucleic acid amplification, the offset temperature was kept slightly higher than the ideal temperature due to the surrounding environmental circumstances. In Figure 8B, T-shot is pointed toward the annealing region which is 60°C (required temperature).
(A) Denaturation (94°C). (B) Annealing (60°C). (C) Extension (72°C).
Gel electrophoresis
Gel electrophoresis is a method of separation based on the size of molecules (including DNA, RNA and proteins) by applying an electrical charge of 100 V via an agarose matrix comprising tiny pores. Here, a 1% agarose gel was prepared in tris-acetate-ethylenediaminetetraacetic acid buffer using a standard protocol [55]. The electric field has two poles, one with a negative charge that pulls molecules and the other with a positive charge that pushes molecules through the gel. The electric field strength was 5 V/cm (100 V/20 cm). SYBR Green-I was used as a highly sensitive dye for visualizing DNA in the agarose. A 20-μl PCR mixture was prepared and infused in the microchannel using an automated syringe pump through an inlet at an optimized flow rate of 5 μl/min, with a total reaction time of 18 min 41 s. Figure 9 shows the amplification results of the non-infectious rat GAPDH gene. After the amplification process, the PCR product was automatically moved to the PMMA-based microchamber between the LED and the photodiode. The PMMA microchamber was created using the CO2 laser ablation technique and was used as a reservoir to collect the sample from the PDMS–glass microfluidic device through pressure-driven force for detection. As mentioned above, in the earlier study by our research group, a standard gel electrophoresis technique was performed for the obtained amplified sample, and it showed promising results.
Lane M: 1 kbp ladder marker; lane 1: PCR product from the microfluidic device.
DNA amplification analysis using on-site detection unit
Herein, a photometric-based detection system was employed with an LED and a photodiode. The absorbance value displayed a linear trend when the cycles were varied, as shown in Figure 10A. The experiment was performed using a conventional PCR instrument for 10, 20 and 40 cycles, whereas 30 cycles were executed on the proposed device, as highlighted in Figure 10A. The obtained absorbance data showed good repeatability of the proposed system, with a correlation coefficient (R2) of 0.9901, signifying excellent linearity. As expected, the output voltage decreased with an increase in absorbance with an increasing number of amplification cycles. Similarly, the absorbance increased with an increase in the concentration of DNA, because the SYBR Green-I used is a cyanine dye added with nucleic acid; the resulting DNA–dye complex best absorbs 497-nm blue light and emits green light at 520 nm. Further, experimentation was conducted for 30 cycles for both the conventional instrument and the proposed device, and these samples were subjected to photometric-based detection, wherein a similar absorbance value was recorded, as shown in the dotted box in Figure 10A. Figure 10B shows the correlation between the absorbance measured with a conventional thermocycler and that obtained with the proposed device. The detection system found no or negligible absorbance for the crude (unamplified) sample. This is a progressive study wherein, we tried to establish a platform for testing nucleic acid samples using microfluidic technology; thus only one PCR was performed on this proposed device, as a proof of concept. The reproducibility of the device was good (three or four iterations were performed on the device). In our earlier study, we reported different detection techniques, such as standard gel electrophoresis and electrochemical methods [41,56].
(A) Results show an increase in absorbance with a concurrent increase in the number of amplification cycles of the PCR (highlighted 30 cycles performed on the proposed device). (b) Correlation between the absorbance as measured on the proposed miniaturized device and on a standard PCR 96-well thermocycler.
Conclusion
The present work describes the development of a fully automated and integrated portable thermal management system with a microfluidic on-site detection unit used for nucleic acid amplification. The heater and sensor were controlled, processed and monitored using an Arduino-based microcontroller chip. The thermal management system was based on the PID controller principle to achieve precise, constant and consistent temperature output. A serpentine-based microfluidic device was fabricated using a soft lithography technique with a master developed by the UV-DLW instrument on a DFR. Two copper blocks were utilized as a heating medium to generate the desired temperature on the microfluidic device with two PCR thermal zones – denaturation (95°C) and annealing (60°C) – and an extension (72°C) zone was obtained by equilibrium and was ensured using an infrared camera which was successfully established on the proposed thermal management system. To perform on-site photometric detection, the device uses a suitable combination of an LED and a photodiode. To improve the signal obtained from the photodiode output while reducing noise, a transimpedance amplifier circuit was designed. The device is incorporated with IoT and Bluetooth modules for a real-time data-logging facility connected to a smartphone, making the device more versatile and amenable to POC testing. A PCR mixture of 20 μl volume with SYBR Green-I dye was infused in the microfluidic device through an automated syringe pump with an optimized flow rate. Here, each time, the absorbance was increased with an increase in the number of amplified cycles. Furthermore, this was compared with that of an unamplified sample to monitor the amplification of nucleic acid on the proposed device integrated with a suitable photodiode LED combination. Herein, as proof of principle, the GAPDH gene (594 bp) was successfully amplified and detected on a single microfluidic device. The overall amplification time was reduced.
Photometric-based on-site detection opens the way for a fully automated and integrated microfluidic device that can be used for several applications in the future to harness microfluidic devices for biological and clinical diagnostic purposes. Further, the microfluidic device is amenable to use for numerous temperature-based processes such as biomedical, biochemical and nanomaterial synthesis applications. Currently, work is going on to develop and integrate an automated and miniaturized fluid injection system for easy infusion of the reaction sample onto the microfluidic device. The device can be fully integrated and automated as a commercial product in the future, allowing it to be used in a wider variety of field-deployable applications.
Author contributions
M Kulkarni: conceptualization, methodology, data curation, validation, analysis, writing (original draft preparation). S Goel: visualization, investigation, supervision, project administration, resources, writing (reviewing and editing).
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Data availability statement
The data that support the findings of this study are available within the article.
Open access
This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
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