Please can you explain what DNAzymes and gold nanoparticles are?
Gold nanoparticles are tiny spherical particles made out of gold atoms with sizes on nanometre scale. This is around 1,000 times smaller than the thickness of human hair.
One important feature of gold nanoparticles is that they appear very brightly red in colour, making them optimal components for assays that generate results that can be visualized by a naked eye. For this reason gold nanoparticles are used to generate the red colour on the common pregnancy test strips.
DNAzyme is an enzyme made completely out of DNA. DNAzyme has the ability to cleave another “substrate” DNA strand into two halves, effectively acting like a pair of molecular scissors.
A single DNAzyme can sequentially cleave multiple substrates. Just imagine walking around with a pair of really tiny scissors and cutting one DNA strand after another.
How have DNAzymes and gold nanoparticles been used to test for infectious diseases?
Another property of gold nanoparticles is that if they are clumped together, which puts nanoparticles in close vicinity of each other, the electromagnetic fields of individual particles interact. The result is the change in gold nanoparticle colour from red to purple.
For our test we developed a network of gold nanoparticles linked together by multiple DNA strands. In this network the nanoparticles are clumped, and therefore appear purple. The DNAzyme scissors could then be used to disassemble the network by cutting the DNA links holding nanoparticles together.
For this purpose we used a modified variant of DNAzyme, which only becomes active if genetic material associated with a particular infectious pathogen is present. This genetic material, or target, can essentially be thought of as the bolt holding the two parts of DNAzyme scissors together.
Activated DNAzyme then cut the DNA links holding gold nanoparticles together, the clumped network is dispersed, and nanoparticle solution turns red. Therefore, the colour of the solution directly indicates whether the pathogen is present (red, dispersed nanoparticles), or absent (purple, clumped nanoparticles).
Since a single activated DNAzyme can cut multiple DNA links, a lower amount of genetic target is needed to disperse the nanoparticles. This is the signal amplification step that significantly improves the sensitivity of pathogen detection.
How was this test developed?
The test platform was developed by arranging a number of previously reported molecular components into a novel configuration. The technique of assembling gold nanoparticles into a network using DNA strands and using the colour change as the readout was pioneered by Chad Mirkin in Northwestern University.
However, the original implementation did not include any signal amplification steps for low detection capabilities. Similarly, DNAzymes that get activated by the genetic material from pathogens were reported previously, but required expensive equipment to read out the results.
For our test we addressed both limitations simultaneously by combining the two components. DNAzyme provides the signal amplification step that was lacking in gold nanoparticle colorimetric assay.
At the same time, introduction of gold nanoparticles replaces expensive equipment with a simple colour readout of DNAzyme activity. The result was a simple, sensitive and cost-effective assay with potential to be used for point-of-care testing in the developing world.
What infectious diseases can this test diagnose?
By very simple DNA sequence adjustments DNAzyme molecule can be made to respond to any genetic material. Therefore, any pathogenic disease can potentially be detected using our approach. In real life implementation, however, the assay use might be limited by the amount and ease with which genetic material can be extracted from particular pathogen.
Our test is currently in the development stage, so further experiments will need to be performed to better identify applicability to different infections in clinical setting. To date, we have been able to detect genetic signatures from malarial parasite, bacteria causing gonorrhea and syphilis infections, and hepatitis B virus.
How does this test differ from more traditional tests?
There are two main types of traditional tests for the detection of infectious disease based on their genetic material signature. The first type includes highly sensitive tests such as polymerase chain reaction, or PCR.
The main advantage of these tests is strong enzymatic signal amplification. Therefore, even when a very small amount of genetic material is initially present, the test will be able to detect it. These tests, however, require very expensive equipment and trained personnel, limiting their use to well-established clinical laboratories.
The second set is best represented by dipstick format similar to the pregnancy test, or a direct aggregation of gold nanoparticles by the genetic material. While these test formats are very simple and provide colour readout of the results, they generally lack any signal amplification steps, and therefore have low sensitivity.
In our test we combine the colour detection benefits of gold nanoparticles, with signal amplification introduced by DNAzyme scissors-like activity. Therefore, our system includes both the simplicity and portability of pregnancy-type tests, and improved sensitivity that is characteristic of the clinical assays that include enzymatic amplification step.
What benefits are there of this test?
There are a number of benefits. First of all, intense red and purple colours associated with gold nanoparticles allow the assay results to be read by a naked eye without the need for any expensive and complicated equipment.
Secondly, introduction of DNAzyme as the means of signal amplification significantly improves the sensitivity of genetic material detection. In addition, we have been able to convert all of the assay molecular components into powder. When compared to liquid solutions, the powdered components are much more stable and lower in weight and volume, allowing their long-term storage and ease of transport.
Finally, the test uses very cost effective all-DNA components. Similarly, gold nanoparticle production requires very little gold to be used due to their small size and can be easily manufactured on very large scale.
The test is fast, very simply to perform, and can detect multiple diseases in parallel. Taken together, these factors make our test particularly suitable for point-of-care applications in the developing world.
Why is it important to be able to identify dangerous infectious diseases rapidly?
Infectious diseases are the main cause of death in the developing world. As such, they result in very significant losses to human life and global economy. Whenever an infectious outbreak occurs, it is essential to contain it as soon as possible to minimize its spread.
However, the only way to really achieve this is to provide doctors with the ability to accurately and rapidly detect infections in remote locations. If needed, the patient can then be treated on the spot and quarantine initiated to prevent further spread of the disease.
Naturally, it is much preferable if this kit can test for multiple diseases simultaneously. If, on the other hand, patient samples first need to be sent to a central diagnostics centre for identification, critical time is lost. Therefore, ability to perform rapid point-of-care testing might mean the difference between a localized outbreak, which is quickly contained, and a region-wide pandemic.
Are there any limitations of the test?
While we have demonstrated the benefits of DNAzyme-gold nanoparticle system in detecting multiple pathogenic targets in laboratory setting, significant work still needs to be done to develop a fully functional kit that a medical professional could use in the field.
One important aspect is the extraction of genetic material from the pathogens. A number of simple methods have been reported that can achieve this, but we still need to identify the best one and to adapt it for our kit.
Secondly, while we demonstrated a significant improvement in sensitivity, it is still not high enough to properly detect many of the infectious diseases. Therefore, there is a need to further improve signal amplification. One of the approaches is to adapt the DNAzyme signal amplification strategy recently developed by Itamar Willner’s group in The Hebrew University of Jerusalem.
What impact do you think the test will have?
Currently, a large number of tests are being reported as alternative of the highly sensitive PCR strategy. They often use very innovative approaches to achieve sensitivities close to that of the PCR. However, we think that it is important to keep in mind that in a clinical laboratory setting, PCR for diagnostics is nearly perfect. In some cases it can detect the signature of just a single organism. As a result, PCR has become the method of choice and is unlikely to be replaced in the near future.
However, outside the clinical laboratory PCR test becomes too complex and expensive, and that is the niche for which new tests need to be developed. In this context, we believe that there is currently too much emphasis on very high sensitivity, and not enough on requirements that make the test applicable outside of the lab. These include low cost and simplicity, stability and transportability of the components, parallel detection of multiple targets.
Nevertheless, sensitivity remains an important requirement. Therefore, it is imperative to introduce a signal amplification step that does not contradict the other requirements for the cost, stability and simplicity. We designed our test to fulfil these requirements. We believe that our results will prompt other researchers to follow similar guidelines when designing their own diagnostics tests.
Where can readers find more information?
Please read our manuscripts on DNAzyme and Nanotechnology based diagnostics. The references are Zagorovsky, K. and Chan, W. C. W. (2013) Angewandte Chemie, 52, 3168 and Hauck, T. S., Giri, S., Gao, Y., Chan, W. C. W. (2010) Advanced Drug Delivery, 62, 438.
About Dr Chan and Kyryl Zagorovsky
Professor Warren C. W. Chan is currently a Full Professor at the University of Toronto at the Institute of Biomaterials and Biomedical Engineering and Donnelly Centre for Cellular and Biomolecular Research.
His lab is focused on the development of nanotechnology for cancer and infectious disease applications.
He has won the Rank Prize Fund (UK), International Dennis Gabor Award (Hungary), and NSERC Memorial Steacie Fellowship. The attached picture is taken by members of NSERC.
Kyryl Zagorovsky completed his undergraduate degree in the University of Toronto specializing in the Astrophysics and Microbiology disciplines.
He is now a Ph.D. student in the laboratory of Professor Warren Chan in the Institute of Biomaterials and Biomedical Engineering in the University of Toronto.
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