Ultrafast Real-Time PCR in Photothermal Microparticles

Abstract

As the turnaround time of diagnosis becomes important, there is an increasing demand for rapid, point-of-care testing (POCT) based on polymerase chain reaction (PCR), the most reliable diagnostic tool. Although optical components in real-time PCR (qPCR) have quickly become compact and economical, conventional PCR instruments still require bulky thermal systems, making it difficult to meet emerging needs. Photonic PCR, which utilizes photothermal nanomaterials as heating elements, is a promising platform for POCT as it reduces power consumption and process time. Here, we develop a photonic qPCR platform using hydrogel microparticles. Microparticles consisting of hydrogel matrixes containing photothermal nanomaterials and primers are dubbed photothermal primer-immobilized networks (pPINs). Reduced graphene oxide is selected as the most suitable photothermal nanomaterial to generate heat in pPIN due to its superior light-to-heat conversion efficiency. The photothermal reaction volume of 100 nL (predefined by the pPIN dimensions) provides fast heating and cooling rates of 22.0 ± 3.0 and 23.5 ± 2.6 °C s^-1, respectively, enabling ultrafast qPCR within 5 min only with optical components. The microparticle-based photonic qPCR facilitates multiplex assays by loading multiple encoded pPIN microparticles in a single reaction. As a proof of concept, four-plex pPIN qPCR for bacterial discrimination are successfully demonstrated.

Keywords: bacteria; hydrogel; multiplex assay; photonic PCR; real-time PCR; reduced graphene oxide (rGO).

Figure 1

Schematic overview of pPIN qPCR. The microparticles in a plastic chip were irradiated to generate heat, and their fluorescent signals were measured in each cycle. Laser power is controlled for thermal cycling via temperature feedback from a thermal camera. (a) Microscope image of encoded pPINs. (b) Cryogenic SEM image of cross-section of pPIN. (c) pPIN qPCR system configuration. (d) Schematic of the photonic reaction in hydrogel matrix of pPIN.

Figure 2

(a) Absorbance measurement of photothermal nanoparticles in DI water. (b) Photothermal heat generation curves of photothermal nanoparticles in DI water under the irradiation of an 1 W 800 nm laser. (c) Photothermal heat generation curves of pPIN under the irradiation of an 1 W 800 nm laser. (d) Temperature profiles with respect to the volume of the pPIN under same light dose of 0.28 W. Dash line showed the temperature profile of 100 nL of pPIN triggered by the irradiation with a light dose of 1.3 W.

Figure 3

(a) Schematic for photonic PCR of the rGO embedded pPIN. Primers adsorbed on rGO surface are released when PCR starts, and target DNA is amplified within the pPIN microparticle through photonic cycling. (b) Photothermal heat generation of pPINs made with various rGO concentrations. Fluorescent signals of (c) amplification and (d) its normalized values obtained with Peltier-based thermal cycler according to various rGO concentrations in pPIN. (e) Fluorescent images of the pPINs at the first and the last cycles on Peltier-based thermal cycler with varying concentrations of rGO. Concentration of rGO denotes the value after mixing with the primer (1:1, v/v).

Figure 4

(a) Temperature profile for 40-cycle PCR. (b) Temperature stability on denaturation (black) and annealing/extension (red) steps. (c) Heating and (d) cooling rates during 40-cycle PCR.

Figure 5

Amplification performance of singleplex pPIN qPCR for E. coli. (a) Serial snapshots of pPIN qPCR for targeting E. coli 10⁷ copies μL^–1. (b) Ten-fold serial dilution of pPIN qPCR for targeting E. coli from 10⁸ to 10² copies μL^–1. NTC denotes no-template control. (c) Standard curve of pPIN qPCR for targeting E. coli.

Figure 6

Ultrafast pPIN qPCR with 10⁷ copies μL^–1 of E. coli. (a) Temperature profile and normalized amplification curves of 40-cycle ultrafast pPIN qPCR with varied duration time. (b) Serial snapshots of 5 min ultrafast pPIN qPCR with or without the DNA template (PTC and NTC).

Figure 7

Multiplex pPIN qPCR for bacterial discrimination. (a) Codes of pPINs for each bacterial target. Images and amplification curves of multiplex pPIN qPCR with the introduction of (b) E. coli, (c) K. pneumoniae, (d) P.aeruginosa, and (e) A. baumannii (the first and 40th cycles).