Introduction

Before developing nucleic acid (NA) amplification based diagnostic method in 19831, humanity experienced significant losses due to pandemics of infectious diseases caused by viruses, bacteria, and fungi2. In fact, pandemics, such as the Spanish Flu3, Asian Flu4, Seventh Cholera5, and Hong Kong Flu6 reached the global death toll of more than 1 million because slow and inaccurate serological diagnostics7 hindered effective containment. However, post-1983, the casualties of severe acute respiratory syndrome (SARS)8, influenza9, and Middle East respiratory syndrome (MERS)10 pandemics in 2002, 2009, and 2012, respectively, were under 200,000 as shown in Fig. 1, because of developing a polymerase chain reaction (PCR) which amplifies NAs by repeating a temperature cycle and is a standard method for molecular diagnosis that can accurately detect small amounts of NAs1. However, even under PCR era, COVID-19 causing 777 million cases, 7 million deaths, and major social and economic impact worldwide11 during the last six years because of their high transmissibility in modern society where the prompt containment of infection is highly demanded based on quick and accurate diagnosis by individuals. Consequently, faster, cheaper, more sensitive, and more accurate NA amplification-based point-of-care testing (PoCT) platforms have been demanded globally12,13,14,15 to control viral spread in the early stages of emerging infectious diseases.

Fig. 1
figure 1

Pandemic death toll. Graph showing the number of deaths from infectious diseases that have been prevalent worldwide since the 20th century. Looking at before and after the time of PCR development, the number of deaths from infectious diseases has decreased significantly since 1983 when PCR was developed. These statistics show that the spread of infectious diseases can be effectively controlled through rapid and accurate diagnosis of infectious diseases

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While passing through the COVID-19 pandemic, worldwide research for developing NA amplification-based rapid and accurate PoCT methods has been intensively carried out16. Especially, isothermal amplification methods, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have been intensively investigated for developing NA amplification-based PoCT because they can amplify NA without bulky and complex thermal cyclers of PCR, but only requires a simple thermal control system to keep isothermal temperatures of 65 °C17 and 37 °C18, respectively. However, the isothermal amplification methods generally result in higher non-specific amplification19,20 than PCR, resulting in false positives. In addition, LAMP requires six primers, which poses significant challenges for multiplexed detection because of the potential primer-dimer formation and increased analytical complexity. Furthermore, both LAMP and RPA have limited quantification because of unclear correlation between color change and NA concentration because of colorimetric reading method which is highly dependent on individual’s interpretation of the color intensity. Therefore, the PCR will be a good candidate for developing NA amplification-based PoCT with 6S: simplicity, speed, small, sustainability, sensitivity, and specificity.

To diagnose the infectious diseases by PCR-based PoCT with 6S, sample preparation and injection should be simple to use by untrained personnels, speedy thermal cyclers should be equipped for the rapid NA amplification, and the size of PCR-based PoCT should be small enough to carry, while maintaining sensitivity and specificity. In addition, the sustainability in manufacturing disposable parts of the PCR-based PoCT and landfills of them should be considered as well. However, up to present, the PCR-based PoCT which can meet 6S has not been reported yet. Thus, this review presents a guide for the practical development of the PCR-based PoCT with 6S, called QUICK-PCR: quick, ubiquitous, integrated, and cost-efficient molecular diagnostic kit based on PCR system.

To develop the prospective QUICK-PCR, this review first analyzes main fundamental elements in a PCR diagnostic system: the sample preparation process, thermal cycler system, and result readout system. Based on the analyzed main elements, innovative technologies in the main elements to meet 6S were briefly introduced and comparatively summarized key technologies to implement them in developing the QUICK-PCR and analyzed the disposable parts with the view of the sustainable manufacturing system including their landfills. In addition, the review considered the clinical validity of any innovative technologies and parts in the PCR-based PoCT which will be shrunk the size and fabricate with a high-throughput additive manufacturing method to meet simplicity, speed, small, and sustainability while compromising the sensitivity and specificity to meet the regulations21 in FDA and CE-IVD. As such, the prospective QUICK-PCR by addressing a way of resolving 6S and issues of clinical validation can practically guide researchers to develop rapid, cost-effective, and universally accessible diagnostics for serving as an efficient tool for future pandemic response and other public health crises.

Polymerase chain reaction: gold standard molecular diagnosis method

Although during the COVID-19 pandemic, a substantial number of publications emerged on molecular diagnostics-based PoCT using methods such as PCR, LAMP, and RPA, only PCR has been clinically proven accurate through many technological advances and numerous clinical experiments22,23,24, and international standards and regulatory systems are well established25,26 for global use. Therefore, it is used as the gold standard of molecular diagnosis. Thus, we provide a comprehensive overview of RT-PCR technology in this section.

The standard diagnostic process for PCR consists of four steps27 respectively, by sample collection, sample preparation, thermal cycling, and fluorescence detection. In the case of COVID-19, a nasopharyngeal swab is used to collect target NAs from a patient (Fig. 2a). The extraction of target NAs from the collected swab involves several key steps as shown in Fig. 2b. In general, the cell membranes are first lysed, and then, contaminants are removed via chemicals such as phenol or chloroform, or enzymatic treatment by using silica-based columns. After the pre-treatment, the purified target NAs are retrieved by centrifuging the treated solution from the previous stage. After injecting purified target NAs, the thermal cycling of the PCR consists of three main temperature steps (Fig. 2c): the first step is denaturation, which occurs in the temperature range of 93–95 °C and is the process of separating the double strand DNA to a single strands. The second step is annealing, which depends on the melting temperature of the primer, and usually occurs in the temperature range of 55–65 °C. This is the process of attaching forward and reverse primers, which have a nucleotide sequence specific to the target amplicon to the 5’-end of each single strand DNA. As the third step, elongation step begins at 72 °C, where the polymerase and dNTP react to replicating the target amplicon sequence, starting from the primer-attached position. For RNA amplification, complementary DNA (cDNA) is synthesized from RNA before starting the thermal cycles using an isothermal reverse transcription step with the help of reverse transcriptase. Through this process, a small amount of the initial target NA is amplified by a factor of 2n (where n is the number of PCR cycles), facilitating the more accurate detection and analysis of the target NAs. As a readout step, the amplified DNA can be analyzed using three different methods (Fig. 2d): 1. Gel electrophoresis28, 2. real-time amplification curves29 and 3. end-point detection30. Based-on these fundamental three steps in PCR, it can be categorized into three generations according to the result analysis methods, as follows:

Fig. 2
figure 2

Overview of the PCR-based molecular diagnostic process. a Sample collection: Biological samples were collected from patients using nasopharyngeal swabs. b Sample preparation: Collected samples release NA through lysis, and RNA is then extracted and purify RNA using column-based methods. c RT-PCR amplification: Extracted RNA was converted to cDNA through reverse transcription and then subjected to thermal cycling for target gene amplification. d Detection: Amplified DNA is analyzed using gel electrophoresis, real-time fluorescence detection, or digital PCR

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The first-generation PCR, conventional PCR method is used gel electrophoresis28 to detect DNA after thermal cycling. Gel electrophoresis is effective in separating DNA fragments by size, but it is difficult to distinguish minute variations due to the low resolution between fragments of similar size, and errors in experimental results may occur because they are sensitive to gel concentration and applied voltage. In addition, the experimental process is prolonged, which provides information about presence or absence of DNA but not accurate quantitative analysis. Moreover, gel electrophoresis is performed on separate equipment rather than on a thermal cycler requiring transfer of the amplified solution which can result in cross-contamination.

Real-time PCR or qPCR alleviates the problem of cross-contamination by eliminating the additional process for gel electrophoresis and replacing it with a real-time analysis method29, thereby simplifying the detection process. qPCR uses fluorescence signals to quantify DNA in real-time during thermal cycles using dye-based31 (e.g., SYBR Green) and probe-based32 (e.g., TaqMan) approaches. Furthermore, multiplex detection is achievable for diagnosing various infectious diseases using a single PCR by attaching different types of fluorophores to the end of the probe33. Despite increasing the feasibility of PoCT by replacing bulky optical reading systems with smartphone LED and camera34,35, qPCR still relies on standard curves and is sensitive to inhibitors and contamination.

Digital PCR (dPCR) enhances the limit of detection (LoD) and more reliable sensitivity for the low-abundance targets by partitioning samples into numerous individual PCR reactions (microwell plates, oil emulsions, and microwell chips), allowing the absolute quantification of NA at very low concentrations36. Individual partitions dPCR effectively increases the local concentration of the target within each reaction volume and reduces inhibition and contamination. After thermal cycling, Poisson statistics were used to analyze the number of positive partitions to accurately estimate the number of NA copies37. Droplet-based dPCR (ddPCR)38 and chip-based dPCR (cdPCR)39 are two dPCR methods that utilize microfluidic chips. However, the PoCT application of dPCR faces challenges, such as thermal conductivity fluctuations due to droplet formation40, which require adequate ramping rates to minimize temperature differences. It also requires a droplet generator to generate uniform droplets, increases the complexity and time requirements. Moreover, cdPCR mitigates the issues of ramp rate and eliminates droplet generation, thereby shortening partitioning time and facilitating multiplexing.

Limitations in recent PCR technology

Advances in PCR technology have improved accuracy, thermal cycling speed, and diagnostic efficiency, but it is still difficult to implement practical QUICK-PCR because of the complex operation process, long sample-to-answer turnaround time, and the limitation that bulky equipment is required for result analysis. Typical sample preparation to obtain purified NAs takes about 30 min through a complex multi-step process by skilled personnel. These conditions limit the applicability of PCR in field diagnosis, where simple, rapid, cost-effective, and easy use are required.

To realize QUICK-PCR, current PCR methods should address three major limitations while maintaining its diagnostic accuracy and sensitivity. First, sample preparation should be simplified using microfluidic chip-based implementations which can be used in the field by a general population. Second, diagnostic speed should be improved by improving thermal cycling methods while keeping them portable and low powered. Finally, readout system should be simplified by implementing smartphone-based fluorescence, colorimetric reader or implementing electrochemical based methods eliminating bulky optical systems.

Strategy for realizing QUICK-PCR

Sample preparation methods for ubiquitous access to diagnostics

To diagnose the infectious diseases by individuals, PCR-based PoCT should be simple to use and rapid to check out the results, while maintaining accuracy. Recently, methods for easy and quick extraction of NAs from bodily fluids such as saliva41,42, blood43,44,45, and urine46,47 with simple workflow by incorporating microfluidic chips which exploits mechanical48 and chemical innovations49, have been developed. These bodily fluids contain a variety of impurities which can interfere the activation of PCR enzymes and inhibit amplification50, thus high-purity NA extraction is required for PCR. As a typical clinical example, sample of whole blood consists of blood cells (~45%) and plasma (~55%), with target NA. Most of the remaining impurities in the blood cells are hemoglobin, heme group, and cell debris which can chelate Mg2+ ions, essential for PCR amplification, inhibiting polymerase activity51, and cause non-specific binding, thereby significantly reducing the amplification efficiency. Therefore, high-purity plasma separation should be performed with easy-of-use format for a high-efficiency and accurate PCR reaction. This section discusses the development of a user-friendly sample preparation system with high-efficiency and implementation strategies of a field-type system, and a comparative analysis is presented in Table 1.

Table 1 Comparative analysis of different sample preparation methods
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Microfluidic chip-based sample preparation methods

Microfluidic chips utilize micro-sized channels to direct the fluid flow enabling various methods such as mixing, filtration, transport in compact and automated device52. A typical application involves cell lysis, NA extraction, purification from whole blood and saliva using microfluidic chips minimizing human error and sample processing time in the field. Using an integrated microfluidic device, the sample preparation process can be simplified to enable rapid quantitative diagnosis by extracting NAs from whole blood samples. A demonstration of self-powered integrated microfluidic PoC low-cost enabling (SIMPLE) chip applies the principle of separating plasma from whole blood using a trench structure (Fig. 3a) and incorporates a vacuum battery to maintain fluid flow without an external pump or power source53. Plasma separation was completed with an extraction efficiency of 95% in 12 min from 100 μL of whole blood. Likewise, plasma separation was completed with 100% extraction efficiency within 10 min from 5 μL of whole blood using degas-driven flow and trench filter structures54. Another group succeeded in plasma separation in 4 min from whole blood at a 10 μL capacity using gravity and dielectrophoresis techniques55, but the plasma separation efficiency was reduced to 44% while shortening the time. The vacuum battery method used in these devices can effectively remove air in the microfluidic chip, but over time, air is replenished through porous PDMS block, which can reduce the temperature uniformity and accuracy of fluorescence signals. In addition, microfluidic chips have difficulty controlling the fluid precisely, and a complex design is required to implement precise chip functions, which increases production costs because of the complex manufacturing process.

Fig. 3
figure 3

Various sample preparation methods enable ubiquitous access to diagnostics. The techniques include a µ-fluidic automation adapted from ref. 53, b paper-based extraction adapted from ref. 57, c centrifugal µ-fluidic, reprinted with permission from ref. 45, Elsevier, d ultrasonic lysis, reprinted with permission from ref. 44, Elsevier, e magnetic bead separation, adapted from ref. 65 under a Creative Commons license CC BY 4.0. and f direct PCR, adapted from ref. 66, under a Creative Commons license CC BY 4.0. Each method is optimized for PoC applications. These methods aim to simplify and expedite sample processing, making diagnostic tests accessible in diverse settings

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By sequentially processing cell lysis, NA extraction, and purification using the capillary force of paper-based microfluidic systems, we can implement a cost-effective, disposable, and portable sample pretreatment system by moving samples to a series of areas containing reagents56. An easy application to a variety of biological samples at a small capacity without the need for an external pump or power source, the paper-based platform provides an ideal solution for on-site diagnosis in environments with limited human and material resources. Tang, R. et al. developed a device for extracting DNA by automatically inducing reagents and samples using sponge-based buffer reservoirs, paper-based valves, and channels of various lengths were developed (Fig. 3b)57. The device can extract DNA from 30 µL of whole blood, serum, saliva, sputum, or bacterial suspension within 2 min. Kim et al. completed plasma separation, lysis, and NA purification in 30 min by injecting 3 mL of whole blood into an acrylic-based microfluidic device derived by finger-actuator58. Upon implementation of cartridge body using biodegradable paper and polymers, an environmentally friendly field diagnosis system can be developed, thereby it can be realizing a sustainable QUICK-PCR platform.

The disc-type microfluidic system (Lab-on-a-disc, LoaD) can implement plasma separation, cell lysis, NA extraction, and reagent mixing in a single chip by automating liquid movement along microchannels using centrifugal force. Centrifugation is a widely used method59 for separating plasma from whole blood by separating components in a mixture according to differences in density. When whole blood is placed in a centrifuge and rotated quickly, a centrifugal force acts and heavier components, such as red blood cells, white blood cells, and platelets, are pushed down to the bottom of the test tube, and the low-density plasma remains on top, thereby obtaining high-purity plasma. These systems provide consistency and accuracy in diagnosis by reducing the likelihood of human error and minimizing human intervention. Recently, a cost-effective LoaD-based sample preparation and lysis system has been developed by incorporating roll-to-roll (R2R) printing technology (Fig. 3c)45. This device does not require an external pump or mechanical valve, but a miniature motorized centrifuge. Instead, after injecting 150 µL of whole blood using a photosensitive wax valve, plasma separation, cell lysis, and reagent mixing were continuously performed on one chip to complete NA extraction within 30 min. When using the centrifugation method, reducing the amount of input whole blood can shorten the plasma separation time60,61. Despite these advantages, the dependence on dedicated centrifugation equipment compatible with the chip limits its application in field diagnostic environments.

Other sample preparation methods

Nucleic acids can be extracted by lysing the cell membrane using ultrasonic waves and then separating the NAs using magnetic force. Ultrasound creates cavitation bubbles, and the mechanical force generated when this bubble collapses acts on the cell membrane, lysing the cell membrane and releasing NAs from the cell substrate62. The efficiency of cell lysis depends on the frequency of ultrasonic waves and the solvent composition63, and target NAs are captured from the hemolyzed blood or tissue homogenate using magnetic force and specific binding molecules64. Magnetically bound NAs can be separated using an external magnetic field and eluted to obtain high-purity NAs. This magnetic bead-based method is widely used in NA extraction systems because of its high efficiency and ease of automation, as NAs bound to beads can be effectively separated from unnecessary pollutants. As shown in Fig. 3d44, a NA extraction system was developed using a method in which magnetic beads were dispersed within a supersonic wavelength to generate acoustic pressure. The induced particles concentrated at the background vibration point of a standing wave, and genomic DNA was efficiently extracted within 8 min from 1 µL of whole blood. The oil-immersed lossless total analysis system (OIL-TAS) was combined with a magnetic-based separation method to complete RNA extraction and detection from a 30 μL dose of nasopharyngeal swab specimen in a single system in 30 min65 (Fig. 3e). The OIL-TAS system minimizes the risk of cross-contamination and sample loss by using magnetism to move separated droplets to the next chamber while covered with oil. The system proposes a method to ensure rapid and high reliability for the examination of infectious diseases such as SARS-CoV-2 by efficiently purifying the analyte through magnetic extraction and then performing isothermal amplification to quickly detect NAs.

Taking advantage of the relatively fragile nature of the membrane of a viral pathogen and engineered polymerase, a “direct PCR” method has been developed to perform direct amplification of NAs without a NAs extraction step. Direct PCR, which skips the complicated sample preparation process, has the advantage to simply operate the PCR. In the COVID-19 test, the NA extraction process was omitted, and a direct RT-PCR method (Fig. 3f) using an inert or dissolved sample with heat was introduced66. In this method, after lysis using a surfactant, Triton X-100, mixed with saliva or throat/spinal swab, SARS-CoV-2 was directly detected by RT-PCR. When a high concentration of Triton X-100 (5%) was used, the Ct value slightly increased, and the qPCR fluorescence was slightly reduced, which did not affect the RT-PCR result. This method was clinically verified using COVID-19, resulting in 96% of sensitivity, 99.8% of specificity, and 98.8% of accuracy. Therefore, the method of inactivation using a surfactant may be effectively used for large-scale rapid SARS-CoV-2 screening tests. On the other hand, direct PCR with blood samples67 for the diagnosis of malaria showed 93% clinical sensitivity and 100% clinical specificity using a special enzyme called OmniKlentaq polymerase, which is resistant to PCR inhibitors, and a PCR enhancement cocktail. This result showed high agreement with PCR performed on purified DNA and demonstrated that sufficiently sensitive diagnosis was possible when PCR was performed directly from blood using special enzymes and reagents. In contrast, when the clinical performance of the PCR system was directly evaluated for various respiratory tract infections using nasal swab samples68, it achieved 97.5% sensitivity and 98.6% specificity due to fewer inhibitors compared to blood samples, resulting in 98.6% diagnostic accuracy. This clinical validation supports a practical way in developing the QUICK-PCR, utilizing the direct PCR, since it can eliminate two important barriers, simplicity and disposability in the PCR to transform into the QUICK-PCR. Thus, impurities in running PCR, such as hemoglobin, cell residue, and protein, which inhibit polymerase activity, should be nullified by selecting an engineered polymerase and adding designed molecules or ions. In fact, the inhibitory effect has been reduced by using special reagents, such as an inhibitor resistant polymerase, and optimizing the reaction buffer in clinical tests69,70.

Thermal cycler for quick diagnosis

Commercially available PCR device can accurately and stably control the temperature using a block thermal cycler, but its speed and volume are slow and bulky, respectively, limiting the overall diagnosis speed and miniaturization of the device. To implement a QUICK-PCR system, it is required to design a small thermal cycler and to control the temperature quickly and efficiently. PCR repeats the denaturation, annealing, and extension steps within a specific temperature range. The temperature instability in the annealing/extension step, non-specific amplification can occur71, and the instability of temperature in the denaturation step can cause damage to the reagent72, resulting in reduce the amplification efficiency. This section introduces the latest technologies for realizing fast temperature control using Joule-, thermoelectric-, and plasmonic-based thermal cycling methods, and a summary and comparative analysis are presented in Table 2.

Table 2 Comparative analysis of different heating methods
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Joule heating, also known as resistive or ohmic heating, is a phenomenon in which electrical energy is converted into heat when electrons in a conductor collide with atoms when current passes through it73 (Fig. 4a). The amount of heat generated is directly proportional to the square of the current and the resistance of the conductor. Because energy conversion is performed without an intermediate step or other forms of energy intervention, the theoretical efficiency of Joule heating is approximately 100%74. However, the cooling depends on the surrounding environment temperature and passive cooling mechanisms, such as conduction, convection, and radiation. The local heating effect of Joule heating minimizes thermal inertia and directly heats the microfluidic channel or reaction chamber, resulting in a thermal circulation that is much faster than that of the conventional thermal block. Kim et al. developed a rapid PCR kit that significantly shortened NA amplification time and improved accessibility and efficiency using a Joule heater75. The kit was prepared by combining a nichrome-based thin-film heater with a lateral flow paper strip (Fig. 4b). By achieving a heating rate of up to 16.3 °C/s and a cooling rate of 3.4 °C/s through fast and precise temperature control of Joule heating, SARS-CoV-2 RNA with a volume of 3 µL could be detected within 30 min (Fig. 4c) with 3.36 W of electrical power. In addition, Jeon et al. developed a Ag/carbon fiber film-based resistive heater76, and human coronavirus was detected within 10 min at a heating rate of ~4.5 °C/s with 5 W of electrical power. Although the heat conversion efficiency of the Joule heater is excellent, its use in the PoCT field is limited by technical limitations, such as slow cooling rates due to natural heat dissipation and physical/chemical side effects, such as electrolytic reactions or bubble generation77.

Fig. 4: Various thermal cycling technologies based rapid PCR.
figure 4

a Joule heating, when electrical current passing through a resistive metal generates heat owing to electron collisions (M: metal atoms, e-: free electron). b Joule heater-integrated lateral flow PCR kit, and c 30 cycles of a NiCr thin film-based Joule heater adapted from ref. 75. d Thermoelectric heating uses Peltier-elements, in which, when a voltage is applied, heat is absorbed at the n-type material and dissipated at the p-type material, enabling precise temperature control. e Peltier-element-based thermocycler, and f temperature profiles of Peltier-element-based thermal cycler, reprinted with permission from ref. 79, Elsevier, g Plasmonic heating, when light strikes a metallic nanoparticle, the oscillating electric field of the light induces a collective oscillation of the conduction electrons within the nanoparticle. During the damping process of these oscillations, hot electrons are generated as the energy from the plasmons transfers to the electrons. These hot electrons subsequently lose their energy through electron-phonon interactions, converting it into localized heat, which results in significant temperature increases near the surface of the nanoparticle. h Plasmonic optical wells (POWs) based photothermal conversion effect, and i temperature profiles, reprinted with permission from ref. 84, American Chemical Society

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Another heating method is thermoelectric-based heating using the Peltier effect78, which is based on the phenomenon where thermal difference is generate at the junction when an electric current flows through a structure in which different semiconductor materials (Peltier elements) of n-type and p-type are connected. Cooling occurs by absorbing heat at one junction, and heating occurs at the other junction by releasing heat to create a temperature gradient. Heating or cooling can be precisely controlled using a single element, depending on the direction of the current (Fig. 4d). The Peltier-element-based thermal cycler has excellent precision temperature control and durability and has recently been applied to microfluidic chips to improve the speed and energy efficiency of PoCT devices79 (Fig. 4e). PCR was completed in 130 s at a heating rate of 35.9 ± 2.9 °C/s and a cooling rate of 12.6 ± 0.4 °C/s (Fig. 4f). Peltier-element-based thermal cyclers ensure fast heating and cooling rates but require high electrical power (>30 W) to achieve high thermoelectric efficiency80,81.

Although Peltier-element-based thermal cyclers have been employed in commercial benchtop PCR devices, plasmonic heating methods offer an efficient and innovative approach to thermal cycling by leveraging the localized surface plasmon resonance (LSPR) effect82 of metallic nanostructures, and/or nanoparticles. Light interacts with metallic nanostructures, which induces collective electron oscillations known as plasmon on its localized surface resulting in enhanced electromagnetic field and light absorptions as shown in Fig. 4g. The interaction is highest at the resonant wavelength of the nanostructure because of the mechanism of 1) electron-photon, 2) electron-electron, and 3) electron-phonon interactions which can generate extreme instantaneous heat, called plasmonic heating. By adjusting the light intensity and wavelength, the temperature of the metallic nanostructures can be precisely controlled. In terms of cooling, heat dissipation primarily occurs through natural convection, leading to a slower cooling rate than the heating rate. However, rapid cooling can be achieved by adding an external cooling fan and utilizing the high thermal conductivity of the metal material. Plasmonic heating can reach the target temperature within a few milliseconds and can have high energy efficiency by locally transferring heat to the target region rather than the peripheral part. That’s why the thermal cycler of the PCR can be minimized because complex and bulky heat control unit can be replaced by simple LED units. With the development of nanoparticle engineering and optical control technology83, precision can be improved by uniformly heating the entire sample. Recently, gold nanoparticles (AuNPs) have been introduced into plasmonic optical wells (POWs) based on their plasmonic characteristics. When a resonance laser irradiates the POWs, the gold nanoparticles absorb light and convert it into heat to locally form a thermal gradient inside the reaction chamber84 (Fig. 4h). The device achieved a heating rate of 5.68 °C/s and a cooling rate (Fig. 4i) of 2.61 °C/s, 10 thermal cycles were completed in 264 s. An energy-efficient thermal cycler was developed by achieving a heating rate of 13.84 °C/s and a cooling rate of 8.97 °C/s using low electrical power with a plasmonic heating system using a gold film substrate85.

Integrated design for simple readout

From the perspective of a general user, determination of infection is important than knowing the stage of infection. A sensitive detection of the result is required to accurately and intuitively deliver the result to the user. The LoD threshold can be determined through limit of blank (LoB) analysis using an indicator resulting signal, and quantitative positive and negative judgments can be made based on the threshold86. Conventional PCR detection requires sophisticated equipment such as stable thermal cycler, extensive analysis, and skilled personnel to ensure high accuracy and reliability, limiting the accessibility and scalability of diagnostic methods. Therefore, a simple and reliable reading system in a single system is an essential element of QUICK-PCR. The following section deals with the development strategy of a portable and user-friendly reading system that can provide real-time diagnostic information using optical, colorimetric, and electrochemical technologies, and is summarized in Table 3 of the comparative analysis.

Table 3 Comparative analysis of different detection/readout methods
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The readout system using a smartphone equipped with a high-end camera and processor can be integrated with a dedicated smartphone application to automate image processing and analysis, providing accessibility, portability, and convenience without user intervention34,42. In addition, the accuracy of measurement can be improved by incorporating AI-based algorithms87,88. Recently, a diagnostic platform was developed to detect pathogens such as E. coli O157, Salmonella spp., and S. aureus using a smartphone and a paper-based biochip preloaded with specific primers89 (Fig. 5a). While amplification was performed for 30 min using the LAMP reaction, the fluorescence signal was monitored in real time using a smartphone camera equipped with an imaging dark box and optical elements. The corresponding fluorescence signal is the fluorescence signal emitted when calcein binds to magnesium ions during the LAMP amplification process. The system achieved a LoD of 2.8 × 10−5 ng/μL, and it was possible to identify pathogens at low concentrations, such as 10 CFU/mL, within 4 h in a spiked milk sample. However, the performance of the camera system in smartphone-based diagnostic devices affects the efficiency of the entire system and varies in characteristics such as camera resolution, minimum focal length, sensitivity, and camera placement in the device depending on the type of smartphone; therefore, it should be optimized for each smartphone model.

Fig. 5
figure 5

Simple readout technologies in molecular diagnostics. a A smartphone-based optical detection adapted from ref. 89, b plasmonic cross-linking colorimetric PCR, reprinted with permission from ref. 91, American Chemical Society. c Nanoplasmonic accelerated microfluidic colorimetry adapted from ref. 92, d electrochemical signal analyzer adapted from ref. 97

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The colorimetric detection method, which allows intuitive reading of results with visible color change, uses a pH- or redox reaction detection dye and changes color90 in response to PCR. Hydrogen ions are generated during PCR amplification, which decreases pH value. At this time, the color may change according to the pH change using specific reagents such as phenol red or neutral red. Alternatively, the color change when particles are aggregated or dispersed can be observed using metal nanoparticles. Owing to these color change characteristics, the results can be judged with the naked eye and do not require a separate reading device, making it very useful for PoCT testing. Plasmonic cross-linking-based colorimetric PCR (PPT-ccPCR) is a PCR platform developed to read results with color changes, in addition to the plasmonic-based ultrafast thermal cycling introduced earlier91 (Fig. 5b). Plasmonic magnetic nanoparticle (PMN)-based PCR systems enable rapid temperature changes and efficient PCR amplification. When target DNA is present, a PMN-DNA-AuNP sandwich structure is formed, and capturing the assembly with a magnetic field renders the supernatant colorless. In contrast, in the absence of target DNA, the AuNPs remain dispersed and become red owing to their inherent optical properties, and this color change acts as a clear visual reader. This method showed a low LoD of 5 copies/μL and high detection performance within 40 min. Another colorimetric reading system, the QolorEX platform92 (Fig. 5c), achieved LoD of 4 RNA copies/µL by detecting the color change corresponding to the pH value that changes as NA is amplified. The colorimetric method can quickly and easily check the results, but when it is read with the naked eye, the result value may vary depending on the surrounding environment or time; therefore, by applied an additional reading system, it can be easy and accurate to read the results.

The electrochemical biosensor measures the electrical change in current, voltage, or impedance that occurs when an analyte, such as nucleic acid, cell, bacteria, or protein, is combined with detection probes, such as DNA, peptide, antibody, and aptamer, on the electrode surface93. The main electrochemical reading methods include amperometric94, voltammetric95, and impedance96 methods, which can be ideally used for small diagnostic platforms at a low cost because they enable fast and sensitive detection without relying on optical devices. Recently, a reading system that integrates antigen-capture PCR (AC-PCR), lateral flow assay (LFA), and electrochemical biosensing using a card sized potentiostat directly linked to a smartphone has been developed97. With this system, it is possible to simultaneously detect liver cancer biomarkers such as alpha fetal protein (AFP) and circulating tumor cells (CTCs). First, the target biomarker is amplified through AC-PCR to improve detection sensitivity, and the amplified product can be quickly and easily detected using an electrochemical biosensor (Fig. 5d). As the concentration of AFP or CTCs increased, the electric current increased linearly, providing a precise quantitative analysis of the biomarkers. It showed excellent stability, maintaining 96.2% of the original signal even after repeated tests and storage.

PoCT PCR to transform as QUICK-PCR

By integrating the latest cutting-edge technologies developed through continuous research in the field of sample preparation, thermocyclers, and result readouts, it is possible to implement the existing large, slow, and complex diagnostic devices as small, fast, and easy-to-use PoCT-based diagnostic devices. In this section, the PoCT-type PCR platform and its role in the diagnosis of infectious diseases are summarized, with a comparative analysis presented in Table 4.

Table 4 Comparative analysis of different integrated PoCT PCR devices
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By automatically moving the fluid using convection due to the temperature difference on a toroidal-shaped fluid chip98 and repeating thermal cycling without an external device, as shown in Fig. 6a, the total PCR reaction time was shortened to 30 min through precise temperature control. In addition, the pre-quenched microarray enables high-multiple probe-based result reading using spatial separation and achieves a detection limit of 10 copies/reaction. In addition, single-nucleotide discrimination is possible through innovative probe designs, such as toehold and X-probes, providing very high diagnostic specificity.

Fig. 6
figure 6

PoCT PCR devices integrated with various methods. a Convective PCR in a portable device adapted from ref. 98, b a fully automated microfluidic PCR-array system adapted from ref. 99, c smartphone-operated, handheld dPCR device, adapted from ref. 100, under a Creative Commons license CC BY 4.0. d Palm size plasmonic RT-qPCR device for decentralized molecular diagnostics, adapted from ref. 101, under a Creative Commons license CC BY NC ND 4.0. e R2R-manufactured RT-qPCR device, called MEDIC-PCR, for low-cost, high-throughput molecular diagnostics, adapted from ref. 102, under a Creative Commons license CC BY 4.0

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The Onestart PCR-array system99 is a microfluidic chip that combines sample lysis, NA extraction, and Peltier element-based thermal cycling. It is a small and efficient platform that allows complex diagnostic processes to be performed on a single chip. Because the result analysis is possible in real time with the CMOS camera, it is automated from sample input to result confirmation to minimize user intervention (Fig. 6b). The total turnaround time was 1.5 h, achieving a detection limit of 1 copy/µL. The Onestart PCR-array system is a very efficient and highly utilized platform, as it can diagnose up to 21 pathogens in one run.

With the introduction of the Peltier-element-based thermal cycler and smartphone-based optical detection and remote-control system, a small dPCR system100 was constructed with a size of 100×200×35 mm³ and a weight of ~0.4 kg (Fig. 6c). The system achieved heating and cooling rates of approximately 9.9 °C/min and 8.5 °C/min, respectively; therefore, it took 25 min to amplify the target. Using a PDMS-based microfluidic chip with 4096 partition sections, Poisson statistics were applied at the end of the PCR reaction, enabling the absolute quantification of the DNA replication number without a standard PCR curve. However, issues such as uneven illumination and sample evaporation in PDMS chips have occurred, but image processing algorithms and multilayer chip design can solve these problems.

A palm-sized (190 × 140 × 23 mm³) RT-qPCR device101 (Fig. 6d) combined with a plasmonic thermocycler (PTC), plastic-on-metal (PoM) cartridge, and microlens array fluorescence (MAF) microscope was designed to enable rapid and sensitive detection. First, PTC coats Au nanoislands on the surface of a glass nanopillar array, enabling rapid heating and cooling owing to the high surface area-to-volume ratio. Using a wide range of white LEDs, heating and cooling rates of 18.85 °C/s and 8.89 °C/s were achieved, respectively. Disposable PoM cartridges are capable of rapid heat transfer and optimize the speed of PCR and the efficiency of result interpretation by minimizing signal interference by effectively blocking background light. In addition, precise and stable temperature control was implemented by measuring the temperature in real time using a platinum-resistance temperature detector. The MAF microscope was designed for high-contrast short-range fluorescence imaging, which improved the detection limit by 16 times and the signal-to-noise ratio by 1.33 times using micro-system-based real-time detection. Using this device, SARS-CoV-2 was detected with 87% sensitivity, 95% specificity, and 20 copies/cartridge LoD within 10 min.

As an example of a sustainable diagnostic platform, the PCR chip used in MEDIC-PCR102 was manufactured on a carbon black substrate by mass-production processes, such as R2R gravure and imprinting, at a production rate of 156 PCR chips per minute (Fig. 6e). The carbon black substrate showed high photothermal conversion efficiency, and when irradiated with a 940 nm infrared LED, a heating rate of 22 °C/s and a cooling rate of 2.6 °C/s were achieved, and the entire PCR amplification process was drastically shortened to 15 min. In addition, the diagnostic process was simplified by omitting the complicated NA extraction process using the direct PCR method. Using this platform, they achieved a sensitivity of 94%, specificity of 98%, and diagnostic accuracy of 97% in clinical trials of 192 patients infected with SARS-CoV-2. The MEDIC-PCR is small (62 × 48 × 62 mm³), portable, and enables rapid field diagnosis in urgent situations.

To realize the PCR-based PoCT, the QUICK-PCR, three key words of the PoCT, simple, rapid, and disposable, should be realized in the PCR. The simple test can be attained by using the direct PCR without NA separation steps but usually sacrifices sensitivity as delaying Ct values in PCR curve102. Also, the rapid test can be achieved by using efficient photo-thermal conversion-based PCR system with fast temperature circulation with innovated a bulky thermal cycler into disposable microfluidic chips or cartridges, but since the amplification time is short, it would be difficult to amplify NA with long amplicon sizes103, and the sensitivity will be decreased. However, those microfluidic chips or cartridges can be manufactured via the R2R printing and imprinting in-line continuous high-throughput manufacturing method to reduce the testing cost.

A manufacturing method for fabricating disposable chips or cartridges should consider three major barriers: material cost, disposability, and sustainability. In fact, most commercial diagnostic chips and cartridges use injection molding or hot embossing processes based on thermoplastic resins such as polymethyl methacrylate (PMMA)104, polypropylene (PP)105, polycarbonate (PC)99, and polystyrene (PS)106. This process requires a high temperature of 100 °C or higher and emits a large amount of volatile organic compounds (VoCs)107, raising concerns about environmental sustainability in energy consumption and landfill. In addition, to integrate functional layers in the diagnostic chips or cartridges, a separate fabrication method should be employed, and consequently it raises issues in the energy consumption and process complexities. As an alternative, R2R printing and imprinting integrated high-throughput manufacturing method (R2R-Pr-Im) has been drawn attention to resolve three major barriers in manufacturing disposable parts in the PCR-based PoCT devices. This technology can significantly reduce manufacturing costs via in-line continuous printing functional layers and imprinting microfluidic chips or cartridges on low-cost thin-film substrates such as polyethylene terephthalate (PET)108, cellulose109, and paper110, while resolving the landfill issues by selecting quickly curable PDMS102 and biodegradable resins111,112 under low temperature or UV light113. By selecting inexpensive and biodegradable materials to fabricate the chips and cartridges via the R2R-Pr-Im, the QUICK-PCR can be used to diagnose viral infectious diseases worldwide without landfill problems. In fact, the R2R-PrIm is a very attractive high-throughput manufacturing process in terms of reproducibility, cost-effectiveness, and sustainability for the future QUICK-PCR system. However, the QUICK-PCR may compromise in sensitivity and length of target NAs to provide disposable parts in PCR-based PoCT via the R2R printing and imprinting integrated methods. In addition, the commercialization of QUICK-PCR will require sufficient clinical trials and validation, as well as international standards for plasmonic-based thermal control. To obtain approvals from an agency such as the FDA or CE-IVD, comparable test results must be demonstrated when comparing clinical performance with existing products, and strong clinical evidence such as clinical positive percent agreement (PPA) and negative percent agreement (NPA) of ≥ 95% and LoD of 95% is required21,114. Therefore, new technologies should be carefully selected to integrate as the QUICK-PCR by concerning regulation approval to commercialize.

As a reference to see commercialized products based on a method of NA amplification, Table 5 compares the performance of the currently commercialized products. Most of the products include sample preparation modules, with the RT-PCR method, based on the Peltier-element-based thermal cycler while very few products use isothermal amplification technology or only products that have been approved for Emergency Use Authorization (EUA) during the COVID-19 pandemic. This indirectly shows the gap between technological development and commercialization. As summarized in Table 5, and sufficiently proven technologies must be used for commercialization.

Table 5 Comparative analysis of different FDA-approved molecular diagnostic systems
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Conclusion and future perspectives in QUICK-PCR

In this review, by reviewing three key components of RT-PCR and exploring innovative approaches to these components, we propose QUICK-PCR, as illustrated in Fig. 7, as a next-generation field diagnostic system for managing future pandemics. Leveraging advances in sample preparation, thermocycling, and smartphone-based optical detection, QUICK-PCR can increase its applicability to infectious disease management by accelerating the NA amplification process, increasing diagnostic accessibility, simplifying the process from sample-to-result, and reducing production costs.

Fig. 7
figure 7

QUICK-PCR. a Low-cost QUICK-PCR cartridges mass-produced using R2R gravure and imprinting manufacturing processes. b Conceptual diagram of the QUICK-PCR components, such as the sensor holder, for use in conjunction with smartphones. c Schematic of the QUICK-PCR cartridge. d Implementation of an ultrafast thermocycler using LSPR heating via a smartphone flashlight on a paper-based plasmonic layer integrated into the cartridge. e Sample-to-result process in the workflow of the QUICK-PCR system. It includes direct PCR and plasmonic thermal cycling, which allows samples to be prepared quickly and without extraction, and the detection of the final color change is easy to interpret

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In detail, R2R gravure and imprinting processes make it possible to fabricate a QUICK-PCR cartridge, which is a sustainable, rapid, and accurate diagnostic platform102 (Fig. 7a). The integration of automatic filtration and sample concentration (Fig. 7b, c), plasmonic heating101 with a smartphone (Fig. 7d), and immediate reading function91,115 make the QUICK-PCR platform ideal for use in resource-limited environments. The workflow of QUICK-PCR is as follows (Fig. 7e). 1. QUICK-PCR cartridge insertion: The cartridge in which the reagent required for the PCR reaction is lyophilized is inserted into the holder. 2. Colorimetric detection system calibration: The cartridge has standards for optical color calibration, so that the baseline can be set to accurately analyze the color change of the test sample. 3. Sample injection: When a sample such as blood, saliva, or nasal swab extract is dropped at the inlet of the cartridge, impurities present in the sample can be removed through the filter system built into the cartridge holder to concentrate the target NAs. 4. Alignment of cartridges for plasmonic PCR: The QUICK-PCR cartridge with the plasmonic substrate was inserted into the device, and the position was corrected to align the flashlight and reaction chamber of the smartphone for plasmonic heating. The flashlight of the smartphone controls the thermal cycling by periodically turning it on and off using a separate smartphone application. 5. Read results: The PCR results were analyzed based on color change, and the diagnostic results were displayed in real time through a connected smartphone for immediate reading.

To use the QUICK-PCR platform as an ideal diagnostic platform in a pandemic situation, the following innovative technologies can be combined. 1. Utilizing sustainable production processes: Sustainable manufacturing methods, such as R2R additive manufacturing and eco-friendly materials, can be used to produce a sustainable inspection platform that does not cause environmental pollution, even with the explosive increase in inspection volume during the pandemic. 2. Portability and energy-efficient platform: A highly efficient method of converting input electrical power into heat enables stable operation of the platform with affordable batteries, where the power supply is not smooth. 3. AI and machine learning can improve PCR analysis sensitivity116,117: By introducing AI and machine learning algorithms into QUICK-PCR systems, we can improve the sensitivity, accuracy, and efficiency of diagnosis through a more precise analysis of fluorescent signals and automatic correction of signal interference caused by background noise or air bubbles in the reaction chamber. 4. Integration with digital health platforms: A user-centric design for self-diagnosis and home diagnostics that can provide high accessibility to users.

As shown in Fig. 7 and a brief explanation in implementing technologies, there is no need to develop new technologies to realize QUICK-PCR. However, despite the development of innovative technologies, only a few have been successfully commercialized. This gap between developed novel technologies and commercialization exists because customers require more than proving the concept of technical performance; it demands cost efficiency, consistent quality, regulatory compliance, clinical validation, and an environmentally sustainable production line. These complex requirements act as a significant gap in academically reported technologies in QUICK-PCR to reach the market. In addition, the QUICK-PCR can be expanded to QUICK-ISO for isothermal amplification techniques by overcoming the disadvantages associated with the non-specific amplification of existing LAMP and RPA. With recent advances in such as AI-based analysis and digital LAMP118 and RPA119 platforms, non-specific amplification, can be solved by dividing the reaction into numerous isolated micro-reactions. As this approach minimizes contamination and increases sensitivity and specificity by reducing false positives based on the background noise test, the simplicity of isothermal amplification will be positioned as one of promising candidates for the next generation of the PCR-based PoCT by improving diagnostic accuracy. Given these developments and the possibility of rapid and accurate detection, the QUICK-PCR platform has the potential to strengthen pandemic response and respond quickly and effectively to the threat of new infectious diseases. Continuous investment in research, technology development, and collaboration is essential for the active utilization of these innovative diagnostic tools in the global healthcare system, which will ultimately contribute to protecting public health and humanity from future pandemics.