The nanomechanics revolution in medicine
From antibiotic development to inflammation markers, nanomechanics are bending and shaping the future of the bio-industry. Nanotechnology is still uppermost in the imagination of much of the industry, often seen as something of a Sci-Fi concept. But it is a reality, and it is being used in labs across the world today. Dr Mike Fisher of Bio Nano Consulting describes how it is already producing some remarkable results and innovations.
10 June 2011
A growing problem
Within all areas of medical science, there is a constant need for new techniques, drugs, and ideas. One crucially urgent area is that of anti-bacterial drugs. The current international portfolio of antibiotics is becoming increasingly redundant as healthcare acquired infections (HAI) are on the up, and an increasing proportion of these are with Multi-Drug Resistant (MDR) bacterial strains, which are resistant to normal antibiotics.
For instance, MRSA (methicillin-resistant Staphylococcus aureus) infection rates almost doubled in the five years from 2000 to 2005 in the USA (1). Another resistant strain proving problematic is Vancomycin-resistant Enterococcus (VRE). Because these MDR strains only differ slightly (often by a single mutation) from the common forms of the bacteria, it is feared that with the current rates of antibiotic consumption by both humans and animals, more MDR strains could develop.
However, a novel technique exploiting nanotechnology used in the micro-electronics industry presents the opportunity to speed up the discovery process for antibiotics and other pharmaceutical products, as well as many other diagnostic and therapeutic processes.
The nanotechnology in question uses microcantilevers, and their bending properties to evaluate the binding interactions between small molecules such as drugs or DNA. Microcantilevers are thin (<10µm) strips of silicon (0.5mm long and 0.1mm wide) which can detect the binding of molecules, target analytes, to ligands attached to the cantilever (2).
Each cantilever is ‘functionalised’ with particular surface coatings: these comprise of a primary layer (a self-assembled monolayer, SAM) and ligands that bind to specific target analytes. The ligand is immobilised on one side of the cantilever and the relevant target analyte is added to the system in solution. Binding interactions of the ligand and target analytes results in bending of the cantilever.
These highly sensitive biosensors detect minute changes in surface stress as the drug binds specifically to the ligands tethered on the cantilever (see Figure 1 below). They can also be operated in a dynamic mode, when the added mass of bacteria or other biological substances can be detected.
The full details of how the binding events lead to the compression forces that bend the cantilevers is currently the subject of much debate and interest. In order to exploit the full potential of microcantilevers, it is essential that we have a fundamental understanding of the origins of the surface stress.
One idea is based on the repulsive and attractive forces between positively and negatively charged groups on the molecules that are bound to the functionalised cantilever (3). This raises questions about the effect of the pH of the solution on the results.
Observations suggest stress transduction is a collective phenomenon, since cantilever loading affects the results. Using the Vancomycin study (4) when the faction of the surface covered with ligand, p, is between 0 – 0.1 no nanomechanical signal is detected. The nanomechanical signal increased in an almost linear way between p=0.1 and p=1.
Further evidence that a critical number of binding events are needed comes from the identification of critical percolation thresholds in the system. In the Vancomycin case study, a percolation value of less than 0.075 did not generate detectable bending.
Important characteristics of the nanomechanical cantilevers are the reproducibility, sensitivity and clinical relevance of the results. For the Vancomycin study, reproducibility was high, both within arrays and between arrays. The Vancomycin study also demonstrates that the system is sensitive down to clinically relevant concentrations (3-27µM in this case).
This technology has an obvious application in the race to design a new generation of antibiotics. As described above, VRE is a serious problem; attaching the target molecule of this antibiotic to the cantilevers allows the binding of an antibiotic to the target enzyme molecule to be detected. This means the binding properties of new drugs can be quantified.
A possible development on this could involve using bacteria sources from the patient and infection in question to coat the cantilevers, to see if antibiotic resistance is present. This would help doctors decide which drug to use and save valuable time by not administering ineffective drugs to patients.
These microcantilevers have been used in many other research situations, with fascinating results.
Microcantilevers have been used to detect IFM-induced 1-8U gene expression, implicated in the development of melanomas (since it is involved in the transduction anti-proliferative signals). Complementary single strand DNA was attached to the SAM on the cantilever and RNA from the 1-8U gene expression was in the solution (5). Other RNA was included in the solution to measure the resolution of the system: down to 4 bases pairs of RNA was achieved.
Further down the line, this could be used to detect the response of melanoma patients to anti-interferon therapy. Detecting DTT (an organochlorine insecticide compound) by cantilevers required specific monoclonal antibodies in the solution (6). Building on this achievement, a pesticide contamination detector product could be made available.
Portable instruments capable of analyzing multiple components are becoming available (7) and based on this, ‘point-of-care’ diagnostics is an attractive commercial market. Since the diagnosis and management of complex diseases such as cancer requires quantitative detection of a number of proteins, this is an appealing field for the development of microcantilevers.
PSA (prostate-specific antigen), marker for prostate cancer, can be detected over a wide range of concentrations; from 0.2ng/ml to 60 µg/ml in solution with HAS (human serum albumin) and HP (human plasminogen) at 1mg/ml, making these findings clinically relevant (8). CRP (C-reactive protein, an indicator of inflammation) has also been detected with microcantilevers (9).
The questions of specificity and sensitivity need to be addressed for other disease-related proteins and bio-molecules for the clinical possibilities of this technology to be fully realised.
Advantages for drug discovery
As the above studies illustrate, these Nanomechanical Cantilever Biosensors have clear applications in the drug discovery field. The numerous advantages make it a valuable tool for drug development, but is also very versatile, as the studies above illustrate. One necessity in any experiment, reproducibility, is present in these nanomechanical biosensors.
There is no need to label samples or use external probes, making this new technique a quick single step process for the detection of bio-molecules. The cantilevers can be used in arrays, permitting the screening of multiple drug-target interactions in parallel, saving valuable time in the drug-development pipe-line.
The quality of information provided is high; ligand-receptor binding constants can be quantifiably measured, and with sensitivity down to very low concentrations (100pM), the effects of different antibiotics can be distinguished with differential bending signals of -9±2nm. The nanomechanical signal is not limited by mass and the system can be portable.
Binding can be detected down to very low concentrations, beyond clinically relevant concentrations, giving the system a broad dynamic range. Large quantities of prototype drug molecules do not therefore need to be made to be tested in this system.
The benefit that the microcantilever system offers over standard systems such as SPR (surface Plasmon resonance) is that, in addition to measuring binding constants, it can also measure surface stresses that drugs create when binding to biological surfaces, such as bacterial membranes. This information can be extremely valuable in predicting true in vivo drug efficacy.
As the studies and advantages suggest, there is an obvious application of this technology in the commercial market. It is available on a commercial basis for the assessment of drug-target interactions for a broad range of antibiotics and drugs under development. Bio Nano Consulting have already used it in collaborative work with Targanta Therapeutics of Cambridge MA (now part of The Medicines Company) to elucidate the mechanism of action of its drug orytavancin.
It is hoped that this novel technique can be used in the drug development pipeline to speed up the process of developing much needed antibiotics and other pharmaceutical products, as well as helping bring to clinic and the commercial market a host of new diagnostic and therapeutic techniques, from insecticide detection to cancerous cell markers.
Dr Mike Fisher ,
,Business Developmnet Director, Bio Nano Consulting.
In addition to his role at Bio Nano Consulting, Dr Mike Fisher is the Theme Manager for Healthcare & Life Sciences within the UK’s Nanotechnology Knowledge Transfer Network, an advisor to Barrack Hill Partners, an executive search and HR consulting firm based in Boston and Florida, and a Producer of TechNation/BioTechNation, a technology-based radio show on National Public Radio in the USA.
1. Elixhauser, A., Steiner, C. 2007. Infections with Methicillin-Resistant Staphylococcus Aureus (MRSA) in US Hospitals, 1993-2005, HCUP Statistical Brief #35, Agency for Healthcare Research andQuality, Rockville, MD.
2. Fritz J, et al. 2000. Translating biomolecular recognition into nanomechanics. Science, (288):316-318.
3. Watari M, et al. 2007. Investigating the molecular mechanisms of in-plane mechanochemistry on cantilever arrays, Journal of the American Chemistry Society, (129):601-609.
4. Ndieyira J. W. et al. 2008. Nanomechanical detection of antibiotic-mucopeptide binding in a model for superbug drug resistance. Nature Nanotechnolg, 3:691-696.
5. Zhang et al. 2006. Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA. Nature Nanotechnology 1:214 - 220.
6. Alvarez et al. 2003. Development of nanomechanical biosensors for detection of the pesticide DDT. Biosensors and Bioelectronics. 18(5-6):649-653.
7. Jianrong et al. 2004. Nanotechnology and Biosensors. Biotechnology Advances, 22(7):505-518.
8. Wu et al. 2001. Bioassay of prostate-specific antigen (PSA) using microcantilevers. Nature Biotechnology, 19:856-860.
9. Wook Wee et al. Novel electrical detection of label-free disease marker proteins using piezoresistive self-sensing micro-cantilevers. Biosensors and Bioelectronics, 20(10):1932-1938.