This article was updated on the 3rd October 2019.
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Standard methods for the detection of proteins and nucleic acids in biological samples involve using techniques such as:
- Autoradiography: a P32 labeled deoxyribonucleotide triphosphate (dNTP) is incorporated during a polymerase chain reaction (PCR). This DNA strand is radiolabeled to help isolate DNA bands. The problem here is in the use of a harmful radioactive isotope. Another disadvantage is that only one color band is produced from the DNA sample.
- Ethidium Bromide: an intercalating agent used as a nucleic acid strain for electrophoresis. During exposure to UV light, the nucleic acid tag fluoresces and reflects a color to indicate binding to a DNA molecule.
- Silver staining: involves running the PCR products through a polyacrylamide gel to separate the target proteins in a DNA sample.
- Fluorescent dyes: this is an additional method used to detect nucleic acids and proteins. The method involves incorporating a fluorescent dye into a DNA sample during a PCR process. The PCR products are separated and identified using a laser beam that will excite the dye in each isolated PRC product.
The one advantage to the Fluorescent dye method over all other commonly used DNA detection methods is the fact that multiple colors can be detected using a laser beam (i.e., it allows for multiplexing).
Though these standard techniques are still in practice, they can be time consuming with the added issue of sensitivity when it comes to the multiplexed detection of proteins and nucleic acids. Dr. Raj Mutharasan, a lead researcher at Drexel University, has suggested the application of lead zicronate titanate (PZT) to piezoelectric cantilever sensors for heightening the sensitivity of DNA testing. According to Dr Mutharasan and his team, this can also allow for the rapid testing and isolation of harmful cells in a biological sample.
A Standard Cantilever Biosensor
Cantilever sensors are becoming an attractive tool for diagnostics based on their high sensitivity platform and multiplexed detection technique. Being able to use a sensor that can detect multiple target molecules from small biological samples makes this sensor technology a crucial step in studying the detection of diseases such as cancer.
When it comes to malignant tumors, it is already too late to treat the cancer with a maximum success rate. As cancers spread through blood and particularly the lymphatic system in the body, it then becomes important to measure multiple parameters of biological molecules. Hence, the more we know at the molecular and cellular level, the more can be known about the current state of a disease, and more can be predicted about how it will further develop.
Unlike the standard DNA detection methods that involve DNA labeling, a cantilever biosensor platform involves adsorption of biomolecules on a micromechanical layer. As the biomolecules adsorb onto the surface of the cantilever, the reaction causes a decrease in the surface free energy. A differential surface stress is generated between either side of the cantilever beam as a result of adsorption of biomolecules occurring at one side of the cantilever.
For DNA detection, the hybridization that occurs between the target probes changes the intermolecular interactions within a monolayer at one side of the cantilever layer. This further induces surface stress that bends the cantilever bean and initiates a motion (Figure 1). The deflection of the cantilever caused by surface stress change, the range of several nanometres, is measured using a piezoelectric readout.
Figure 1. Schematic diagram of Cantilever deflection. Binding of a molecule of the sensing surface of a cantilever generates surface stress resulting in bending of the structure. Source: Nordström M, et al. SU-8 Cantilevers for Bio/chemical Sensing; Fabrication, Characterisation and Development of Novel Read-out Methods. Sensors 2008; 8: 1595–1612.
Detecting Specific DNA Strands
Single stranded DNA (ssDNA) can be isolated using a cantilever sensor by coating one side of the cantilever surface with gold and applying a thiol linking agent at one end of the DNA. It has been calculated that there is a variation of 30-50 mN/m in surface stress as a result of ssDNA adsorption.
In finer detail, the process of surface stress is based on adsorption of sulfur atoms on the thiol agent attached to one end of the ssDNA that is also bound to the surface of the cantilever. This sulfur atom acts as a receptor for a strand complementary to the DNA sequence in question. By applying this process of DNA isolation, it is then possible to identify non-complementary strands that carry a mutation.
Challenges of Cantilever Sensor DNA Detection Technology
Cantilever sensors require further development to help improve the selectivity performance in these sensors. One way to optimize the selectivity and sensitivity of the cantilever sensor is to refine the immobilization technique for isolating DNA sequences.
Use of a longer and much thinner surface to the cantilever will increase the sensitivity of the sensor, although a larger surface area to the cantilever will allow for a faster detection rate of the target molecule. This will further be important for reagents that are more specific to the target biomolecules (i.e., by using single chain antibody molecules).
- Lange, D., Brand, O., Baltes. (2002). CMOS Cantilever Sensor Systems: Automic-Force Microscopy and Gas Sensing Application. Germany: Springer Science.
- Datar R, et al. Tools for Diagnostics. MRS Bulletin. 2009; 34: 449–454.
- Nordström M, et al. SU-8 Cantilevers for Bio/chemical Sensing; Fabrication, Characterisation and Development of Novel Read-out Methods. Sensors 2008; 8: 1595–1612.
- http://www.uvm.edu/search/ [PowerPoint Presentation on DNA Detection].
- Cheng, L, et al. Using A CMOS-BIOMEMS Cantilever Sensor for Orchid Virus Detection. 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences. October 2–6, 2011, Seattle, Washington, USA.