It seems everybody is focused on using nanostructures for designing new and hopefully better biosensors of the future. However, integrating nanostructures into real world size biosensors is not trivial and that’s where the importance of three recent reports on manipulating nanostructures comes into play. These reports show possible routes for improving biosensor performance by precise control of the nanostructure shape, size and position.
First report in Nature Nanotechnology from Shana O. Kelly of University of Toronto used nanostructured gold microelectrodes to achieve attomolar sensitivity for detecting oligonucleotides. For detecting oligonucleotide she used a label free sensing method developed in her group that uses catalytic reaction between two transition-metal ions, Ru(NH3)63+ and Fe(CN)6 3-. Further enhancement in sensitivity was achieved by using peptide nucleic acids (PNA) that have higher affinity to complementary sequence than DNA/RNA. The key feature of the work is that increasing the surface nanotexture of the microelectrode increased the accessibility of oligonucleotide to bind to their complementary PNA sequence that in turn increased the sensitivity of the biosensor. Moreover, by modulating the nanotexture of their microelectrode the sensor can be tailored for their sensitivity and their linear dynamic range. The next challenge for the sensor will be to detect oligonucleotide in complex biological samples like serum or cell lysates.
Second report though not directly aimed at improving biosensor sensitivity can nonetheless be used for biosensor applications. The report in Nature Nanotechnology from Tel Aviv University describes a method for fabricating highly oriented and aligned aromatic dipeptide nanotubes (ADNTs) using vapor deposition method. The length, thickness and surface density of the ADNTs can be controlled by simply adjusting the deposition parameters. Moreover, the fabrication method can be scaled up to coat large substrates or can be scaled down to design microfluidics channel or micro/nano patterns. ADNTs have already been used for creating metal nanowires and for biosensing and hence an scalable and easy to implement fabrication method will further improve their applications. Since ADNTs dramatically increases the surface area of the substrate hence the “better accessibility” hypothesis implemented in previous report should provide additional handle for improving sensitivity of ADNTs based biosensors.
Third and final report again is not directly aimed at designing biosensors but their applications to biosensing are easy to see. The report in Science from University of Colorado aligns lithographically created polygonal microparticles in liquid crystals. Though a proof-of-principle at this stage, the technology provides a facile method for creating (meta)materials with unique properties by simply aligning nano/micro meter sized particles with exact control over their position, orientation and assembly. Metamaterials and aligned metallic particles are already been used extensively for biosensing and better methods are constantly needed for further improvement in biosensor fabrication and sensitivity.
Gold Nanoparticles: Bigger and Better
Use of gold nanoparticles/nanostructures for biosensing application is exploding and that will necessarily drive need for strict control over the size and shape of these particles. Until now it is easy to get monodisperse gold nanoparticles of diameter of 2-10 nm using sodium borohydride reduction method and 12-50nm using sodium citrate reduction. Now a new paper in JACS is reporting a new method for synthesis of highly monodisperse gold nanoparticles of diameters upto 200nm. Method uses hydroquinone driven reduction of gold ions in presence of seed gold nanoparticles. Gold nanoparticles can be easily modified using thiol polyethylene glycol.
Gold Nanoparticle Based Scanometric Immunoassay
Continuing on the theme of growing gold nanoparticles with electroless gold deposition, Chad Mirkin’s group is reporting using similar approach to further improve the sensitivity of their scanometric multiplexed immunoassay. Scanometric immunoassay method uses traditional sandwich immunoassay where detection antibody is labeled with gold nanoparticles. After binding of detection antibodies, the gold nanoparticles are grown by electroless deposition of silver hence increasing the scattering intensity. To further improve the sensitivity, the silver enhancement step has been replaced by electroless gold deposition. The improved sensitivity comes from larger particle size- 420nm, 1400nm and 2700nm after one, two, and three round of gold deposition. Whereas, for the silver enhancement method the sizes are 100, 270, and 550nm after one, two, and three cycles.
Using gold enhancement method, the sensitivity for PSA (prostrate specific antigen) was 300aM in buffer compared to 30fM using silver enhancement.
Paper Based Biosensors
Whitesides group is reporting on a new, inexpensive handheld spectrophotometer for quantitative measurement of color change of their µPAD (Paper based Analytical Devices) biosensor. Quantitative measure of proteins is important when different level of proteins reflects different diseases. This technology is part of their continuing effort to provide a cost effective, portable, rugged, adaptable, and easy to use diagnostic devices for locations with limited access to physicians or paramedics. Their earlier work on µPAD includes 3-D microfluidic devices using paper and using camera phones for reading and transmitting data from µPAD.
Another report uses good old lateral flow type technology to look for inhibitors of acetylcholine esterase (AChE) inhibitors on paper based biosensor. All the reagents are integrated on the sensor and the response can be read by eye. Several pesticides could be detected in low nanomolar range within 5 minutes and with minimal matrix effect.
Due to localized surface Plasmon resonance, Gold nanoparticles/nanostructures exhibit an absorbance maximum, typically in visible region of ~520-600nm. Wavelength for maximum absorbance is dependent on shape, size and placement nanoparticles as well as the refractive index of its surrounding. Change in absorbance maximum in response to refractive index change has been exploited extensively for biosensing. Now Bengt Kasemo group at Chalmers has used the same property to study the heterogeneous catalytic reaction at the catalyst surface.
The sensor is simple in its implementation- it consist of array of gold nanodisk covered with thin coating of catalyst support surface on which Platinum (Pt) catalyst is deposited. Absorbance spectra of the sensor are collected and shift in maximum wavelength is observed as the catalytic reaction takes place on solid nanometer sized catalyst.
To detect oxidation of hydrogen to water and oxidation of carbon monoxide to carbon dioxide they designed a sensor consisted of an array of gold nanodisk (76nm in diameter and 30nm in height) covered with 10nm of SiO2 on which 5-20nm Pt particles are deposited. Another sensor was created for storage and reduction of NOx by using arrays of gold disks (140nm diameter, 30nm height) coated with thin layer (~30nm) of BaO on which nanogranules of Pt is deposited.
Considering the track record of Prof Kasemo- he is one of the key figures in development of Quartz Crystal Microbalance Biosensor and founder member of the company q-sense- it may not be surprising to see plasmonic sensors hit the market shelves pretty soon.
In a recent post, I compiled a list of commercially available label-free biosensors. The list is fairly impressive with twenty five companies offering label-free sensing based on variety of transduction platform including optical, thermal, mass and electrical. Based on the list, one could assume that market is mature and the space for new sensors will be limited but the furious pace of research in label free sensors presents a completely opposite picture. The question then arises: what is driving the research into new label-free biosensing platforms? My opinion, as someone who has worked in this field for very long time is as follows.
Reason number one: Money, money, money. Bio-sensors in general continue to attract significant funding because of their wide ranging applications in fundamental biological research, clinical diagnostics, environmental monitoring, food testing, and biodefense. The biodefense is one of the key driver for biosensor research especially after 9/11 attack and the anthrax incidents of 2001. Federal funding for Biodefense for Year 2001 was $294 million dollars that jumped to $ 3.1 billion dollar in 2002 and has remained between 5-7 billion dollars since then. A portable biosensor for detection of biological agents will be the key requirements for strengthening the defenses against biological attack at local, state and national level. Clinical diagnostics is another key driver for biosensor research with glucose sensor alone responsible for multibillion dollar market.
Reason number two: Miniaturization and multiplexing and maybe sensitivity. Label-free (bio) sensors are attractive because of single step detection without the need for secondary or tertiary binding event for detection as is required for traditional biosensors and ELISA type of assays. Other attributes (currently available or desired) include, quantitative and sensitive measurement, possibly lower cost as there is no need for reporter labeled detection reagents, single step detection hence simple to use (at least theoretically) and possibly portable. But as of today, commercial label-free biosensors are big laboratory instrumentation, expensive, and difficult to operate. This brings me to my third reason.
Reason number three: Nanotechnology has brought a quantum shift in biosensor research (at least on research scale). Label-free biosensors today are using metal nanoparticles/nano structures, carbon nanotubes, micro/nano electro-mechanical systems (MEMS/NEMS,) nano/micron sized photonic crystals, and lithographically designed micro/nanoscale waveguides for transducing biomolecular binding into optical/electrical/thermal signal. These devices are small, can possible be mass produced at low enough cost and more importantly several of these sensors can be used in parallel for multiplexing. The sensitivity of these sensors are routinely being reported in pM, fM and event attomolar concentration.
I have made a list of key technologies in label-free biosensing that are being actively explored as is evident by several publications in last few years. In addition to brief description of the technology I have included the link to key research laboratories or publications. Publication lists have been picked from Google Scholar Search. These links are by no means only source for research and I will be happy to include more as I gather/get more information. The list is not intended be all inclusive and I expect it to grow over time so come back for updates.
A PDF file of the list with clickable link
Sensitive and multiplexed detection of disease specific protein markers is important for clinical diagnosis. ELISA remains the gold standard for protein detection however the need for multiplexing, higher sensitivity, low sample requirement and capability of point of care testing (POC) is driving the innovations for new biosensing platforms. Changes are coming fast and furious at every level including: a) novel capture agents that include Bio-Mimetic/Antibody-Mimetic Recognition Elements; b) novel surfaces and immobilization methods for oriented and functional immobilization of capture proteins while minimizing non-specific binding; c) new signal transducers like quantum dots, brighter fluorescent dyes, multicolored photonic crystals and up-converting nanoparticles and barcoded nano/micro particles; d) signal amplification methods like rolling circle amplifications and tyramide based amplification; e) new multiplexed sensor platforms like protein arrays, bead arrays and microfluidics and f) label free biosensors based on gold nanoparticles, nanowires, microcantilevers.
To this list can now be added a new biosensor; Magnetic Nanotag Biosensors developed by Shan X Wang group at Stanford. The sensor is a typical sandwich based immunoassay with two critical differences: a) capture antibody is immobilized on a microarray of Giant magnetoresistive (GMR) sensors and b) secondary antibody is labeled with biotin that binds streptavidin tagged with super paramagnetic nanoparticles. Magnetic field induced by paramagnetic nanoparticle is detected by GMR sensors. Concentration as low as 50 attomolar (10-18 M) can be detected using this biosensor and for multiplexing 10 analytes were detected in parallel in quadruplicates (40 reactions). The biosensor is also “Matrix-insensitive” means analyte can be detected with equal sensitivity either from buffer or from complex biological samples like serum.
Though novel and impressive research, I had problem understanding the claim of “Matrix-insensitive protein assay”. Since multiple washings are involved the matrices is long gone before the sensor ever sees paramagnetic nanoparticle for detection. A homogenous assay that is matrix-insensitive makes more sense. Moreover, the problem of auto-fluorescence from biological matrices has been mostly solved by the use of long wavelength fluorescent dyes. Other statement that bothered me is the broad claim that biosensor is 1000 times more sensitive than ELISA. In reality the biosensor performed 1000 fold better for one analyte against one commercially available ELISA kit. It is foreseeable that the commercial ELISA kit used for comparison was the worst kit in the market. Ideally the authors should have created their own ELISA assays (very easy) using the same set of primary and secondary antibodies as used in their biosensor.
Still, a cool technology that avoids the use of any complex optical design and may be ideal for POC devices.