- Fits in the Palm of your Hand, and Has Single-Molecule Sensitivity
- Analyzes 4 Samples, and Can be Modified to do 8
- Project Leader Reveals Commercialization Details
I think we’re all fascinated by catchy headlines touting the world’s biggest, tallest, etc., so a recent publication by Ahrberg et al. in venerable Lab on a Chip claiming the world’s smallest real-time PCR device instantly struck me as blogworthy. It also seemed quite apropos as a follow-up to my previous blogs on the continuing shrinkage, so to speak, of real-time PCR technology for point-of-care qPCR diagnostics or other emerging applications in the field.
This hand-held real-time PCR device, developed by A*STAR Singapore, is amazingly small in comparison to the first real-time PCR system introduced by Applied Biosystems in 1995 that weighed 350 pounds and had a width of 7 feet, thus requiring an entire bench top.
If you’re wondering about performance, this tiny gizmo can perform 40 cycles of real-time PCR detection of four samples in parallel in only ~35 min, and—quite remarkably to me—is capable of detecting a single molecule of DNA in a sample, which is the ultimate goal for limit-of-detection for any molecular analysis.
Luckily this paper by Ahrberg et al. is freely available from Lab on a Chip. However, for your convenience, I’ve attempted to extract and encapsulate some of the details that seem worth sharing here. Interested readers can check out the full paper for more details and the wealth of evolutionary miniaturization by some of the present investigators, and others, discussed in the introduction of this publication.
The system has two key features:
1. Four samples are each contained in virtual reaction chambers (VRCs) on a disposable glass cover slip over micro-machined silicon heaters. Basically, a VCR is an aqueous droplet of PCR master mix and sample covered with mineral oil preventing water evaporation from the sample. In this scenario, the water droplet contained. VCRs are separated from the heaters by a disposable, hydrophobically coated microscope cover slip.
2. Two pairs of 470-nm light-emitting diodes (LEDs) provide fluorescence excitation, and silicon photodiodes are used to detect fluorescence emission in a manner designed to be unaffected by ambient light. The PCR instrument is equipped with a graphical (84 × 48) pixels liquid crystal display (~38 mm diagonal) to show reaction progress in real-time and final results. The captured data is stored in internal memory and can be uploaded for external processing via a standard USB cable. The system is powered by an external 12 V battery.
The real-time PCR protocol uses a fluorogenic intercalating-dye, such as SYBR-Green, and is initiated using a hot start master mix [click here to explore TriLink’s CleanAmp™ hot start reagents which should be compatible on such an instrument]. PCR cycles consist of denaturation, annealing, and extension steps; fluorescence is measured at the end of the extension step for period ≈ 2 s.
Standard samples of a model DNA oligonucleotide target were prepared by adding 12,500 molecules of the hemagglutinin for H7N9 Avian Influenza virus to 200 nL of PCR reaction volume, and then a series of 10-fold dilutions to an average of 1.25 molecules per 200 nL. Data acquired for the 104-bp amplicon was subsequently transferred to a personal computer via a USB interface for further processing, which demonstrated 0.91 ± 0.05 amplification efficiency and single-molecule detection.
The authors conclude by stating that “[t]his tiny real-time PCR device is a promising diagnostic system for remote clinics as well as a tool for educational institution[s] demonstrating the power of a real-time PCR as ‘seeing is believing’. The system throughput can be doubled using a single channel multiplexing method as [we have] demonstrated earlier.”
Interview with Project Leader
I was intrigued by this paper and the thought of a truly hand-held real-time PCR instrument being so close to market, so I reached out to Pavel Neužil (the designated corresponding author for Ahrberg et al) to see if he would provide more details about this project. He graciously agreed to an email interview. Following are Q&A for my (JZ) email interview with Dr. Neuzil (PN).
JZ: Which institute(s) own the patent(s)?
PN: A*STAR Singapore
JZ: Are there plans for commercialization?
JZ: If yes, what year might this device be introduced?
JZ: What is the approximate projected cost (not sales price) for commercial-scale manufacturing of this device?
PN: That greatly depends on the volume, at this point of time it is not that clear.
JZ: Same question but for the disposable, hydrophobically coated microscope cover slips?
PN: We normally coat several dozen or more glasses at a time. Few hundreds can be coated without any problem. The box of 100 glasses cost ~1 USD (retail price) and coating chemicals let’s say 1 USD per batch.
JZ: What is the name/type of the battery used?
PN: The consumption is really low (couple of watts) but we do not use battery but power converter, as that was more convenient at that point of time. It is meant to be powered by car battery or similar readily available power supply.
I hope that you found this blog about “shrinking” real-time PCR instrumentation from 7 feet and 350 pounds to hand held and 1 pound to be as amazing as I did, and would like to share your thoughts as comments here.
In researching Dr. Neužil’s other achievements, I found that he led a team of researchers at the Institute of Bioengineering and Nanotechnology in Singapore that reported in 2006 what was then the world’s fastest real-time PCR device. It utilized a microfabicated chip with a VRC capable of one thermal cycle in 8.5 s, corresponding to 40 cycles of PCR in 5 min and 40 s.
Alas, all records achieved are subject to be beaten, and for speed of PCR it was by Farrar & Wittwer, who reported in 2015 an instrument for “extreme PCR” that amplified 45- to 102-bp targets in single-copy genes from human genomic DNA in 15-60 s—that’s really fast, and quite amazing.
Click here for a video of Carl Wittwer’s very interesting historical overview of his contributions to PCR.