Single-shot multi-channel plasmonic real-time polymerase chain reaction for multi-target point-of-care testing.

Plasmonic nucleic acid amplification tests demand high-throughput and multi-target detection of infectious diseases as well as short turnaround time and small size for point-of-care molecular diagnostics. Here, we report a multi-channel plasmonic real-time reverse-transcription polymerase chain reaction (mpRT-qPCR) assay for ultrafast and on-chip multi-target detection. The mpRT-qPCR system features two pairs of plasmonic thermocyclers for rapid nanostructure-driven amplification and microlens array fluorescence microscopes for in situ multi-color fluorescence quantification. Each channel shows a physical dimension of 32 mm, 75 mm, and 25 mm in width, length, and thickness. The ultrathin microscopes simultaneously capture four different fluorescence images from two PCR chambers of a single cartridge at a single shot exposure per PCR cycle of four different excitation light sources. The experimental results demonstrate a single assay result of high-throughput amplification and multi-target quantification for RNA-dependent RNA polymerase, nucleocapsid, and human ribonuclease P genes in SARS-CoV-2 RNA detection. The mpRT-PCR increases the number of tests four times over the single RT-PCR and exhibits a short detection time of 15 min for the four RT-PCR reactions. This point-of-care molecular diagnostic platform can reduce false negative results in clinical applications of virus detection and decentralize healthcare facilities with limited infrastructure.

Highly Efficient On-Chip Photothermal Cell Lysis for Nucleic Acid Extraction Using Localized Plasmonic Heating of Strongly Absorbing Au Nanoislands.

Cell lysis serves as an essential role in the sample preparation for intracellular material extraction in lab-on-a-chip applications. However, recent microfluidic-based cell lysis chips still face several technical challenges such as reagent removal, complex design, and high fabrication cost. Here, we report highly efficient on-chip photothermal cell lysis for nucleic acid extraction using strongly absorbed plasmonic Au nanoislands (SAP-AuNIs). The highly efficient photothermal cell lysis chip (HEPCL chip) consists of a PDMS microfluidic chamber and densely distributed SAP-AuNIs with large diameters and small nanogaps, allowing for broad-spectrum light absorption. The SAP-AuNIs induce photothermal heat, resulting in a uniform temperature distribution within the chamber and rapidly reaching the target temperature for cell lysis within 30 s. Furthermore, the localized plasmonic heating of SAP-AuNIs expeditiously triggers phase transition and photoporation in the directly contacted lipid bilayer of the cell membrane, resulting in rapid and highly efficient cell lysis. The HEPCL chip successfully lysed 93% of PC9 cells at 90 °C for 90 s without nucleic acid degradation. This on-chip cell lysis offers a new sample preparation platform for integrated point-of-care molecular diagnostics.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Spread spectrum SERS allows label-free detection of attomolar neurotransmitters.

The quantitative label-free detection of neurotransmitters provides critical clues in understanding neurological functions or disorders. However, the identification of neurotransmitters remains challenging for surface-enhanced Raman spectroscopy (SERS) due to the presence of noise. Here, we report spread spectrum SERS (ss-SERS) detection for the rapid quantification of neurotransmitters at the attomolar level by encoding excited light and decoding SERS signals with peak autocorrelation and near-zero cross-correlation. Compared to conventional SERS measurements, the experimental result of ss-SERS shows an exceptional improvement in the signal-to-noise ratio of more than three orders of magnitude, thus achieving a high temporal resolution of over one hundred times. The ss-SERS measurement further allows the attomolar SERS detection of dopamine, serotonin, acetylcholine, γ-aminobutyric acid, and glutamate without Raman reporters. This approach opens up opportunities not only for investigating the early diagnostics of neurological disorders or highly sensitive biomedical SERS applications but also for developing low-cost spectroscopic biosensing applications.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

Nanoplasmonic On-Chip PCR for Rapid Precision Molecular Diagnostics.

Emerging molecular diagnosis requires ultrafast polymerase chain reaction (PCR) on chip for rapid precise detection of infectious diseases in the point-of-care test. Here, we report nanoplasmonic on-chip PCR for rapid precision molecular diagnostics. The nanoplasmonic pillar arrays (NPA) comprise gold nanoislands on the top and sidewall of large-scale glass nanopillar arrays. The nanoplasmonic pillars enhance light absorption of a white light-emitting diode (LED) over the whole visible range due to strong electromagnetic hotspots between the nanoislands. As a result, they effectively induce photothermal heating for ultrafast PCR thermal cycling. The temperature profile of NPA exhibits 30 cycles between 98 and 60 °C for a total of 3 min and 30 s during the cyclic excitation of white LED light. The experimental results also demonstrate the rapid DNA amplification of both 0.1 ng µL-1 of λ-DNA in 20 thermal cycles and 0.1 ng µL-1 of complementary DNA of Middle East respiratory syndrome coronavirus in 30 thermal cycles using a conventional PCR volume of 15 µL. This nanoplasmonic PCR technique provides a new opportunity for rapid precision molecular diagnostics.