Nanotechnology and Microbiology

Dr. Arti Goel, Lecturer, Amity Institute of Microbial Biotechnology, Amity University, Noida, India, recently described the opportunities that nanotechnology has on a wide range of microbiology applications, namely, within the food, clinical diagnostics and water sectors. Here is an excerpt from his white paper, which was recently published on Nanowerk's website (http://www.nanowerk.com/).

Microbiology relates to nanoscience at a number of levels. Many bacterial entities are nano-machines in nature, including molecular motors like flagella and pili. Bacteria also form biofilms by the process of self-assembly (for example the formation of Curli-film by E. coli). The formation of aerial hyphae by bacteria and fungi is also directed by the controlled and ordered assembly of building blocks. Also, the formation of virus capsids is a classical process of molecular recognition and self-assembly at the nano-scale.

Nanotechnology involves creating and manipulating organic and inorganic matter at the nanoscale. It promises to provide the means for designing nanomaterials; materials with tailor-made physical, chemical and biological properties controlled by defined molecular structures and dynamics. The present molecular biology techniques of genetic modification of crops are already forms of what has been termed nanotechnology.

Nanotechnology can provide for the future development of far more precise and effective methods of, and other forms of, manipulation of food polymers and polymeric assemblages to provide tailor-made improvements to food quality and food safety. Nanotechnology promises not only the creation of novel and precisely defined material properties, it also promises that these materials will have self-assembling, self-healing and maintaining properties.

Nanoscience does have an impact on several areas of microbiology. It allows for the study and visualization at the molecular-assembly levels of a process. It facilitates identification of molecular recognition and self-assembly motifs as well as the assessment of these processes. Specifically, there are three areas where microbiologists use nanotechnologists' techniques:

– Imaging single molecules

– Poking and pulling nanoscale objects (laser traps, optical tweezer)

– Determining spatial organization in living microbes (AFM, near/far field microscope).

Nanotechnology in food microbiology

Detection of very small amounts of a chemical contaminant, virus or bacteria in food systems is another potential application of nanotechnology. The exciting possibility of combining biology and nanoscale technology into sensors holds the potential of increased sensitivity and therefore a significantly reduced response-time to sense potential problems.

Nanosensors that are being developed by researchers at both Purdue and Clemson universities use nanoparticles, which can either be tailor-made to fluoresce different colors or, alternatively, be manufactured out of magnetic materials. These nanoparticles can then selectively attach themselves to any number of food pathogens. Employees, using handheld sensors employing either infrared light or magnetic materials, could then note the presence of even minuscule traces of harmful pathogens. The advantage of such a system is that literally hundreds and potentially thousands of nanoparticles can be placed on a single nanosensor to rapidly, accurately and affordably detect the presence of any number of different bacteria and pathogens. A second advantage of nanosensors is that, given their small size, they can gain access into the tiny crevices where the pathogens often hide.

The application of nanotechnologies on the detection of pathogenic organisms in food and the development of nanosensors for food safety is also studied at the Bioanalytical Microsystems and Biosensors Laboratory at Cornell University. The focus of the research performed at Cornell University is on the development of rapid and portable biosensors for the detection of pathogens in the environment, food and for clinical diagnostics. The bioanalytical microsystems use the same biological principles as were used in the simple biosensors, i.e. RNA recognition via DNA/RNA hybridization and liposome amplification. The bioanalytical microsystems that are studied focus on the very rapid detection of pathogens in routine drinking water testing, food analysis, environmental water testing and in clinical diagnostics.

Nanotechnology in medical biology – application of nanodiagnostics in infectious diseases

The rapid and sensitive detection of pathogenic bacteria at the point of care is extremely important. Limitations of most of the conventional diagnostic methods are the lack of ultrasensitivity and delay in getting results. A bioconjugated nanoparticle-based bioassay for in situ pathogen quantification can detect a single bacterium within 20 minutes.

Detection of single-molecule hybridization has been achieved by a hybridization-detection method using multicolor oligonucleotide-functionalized QDs as nanoprobes. In the presence of various target sequences, combinatorial self-assembly of the nanoprobes via independent hybridization reactions leads to the generation of discernible sequence specific detection of multiple relevant sequences.

A spectroscopic assay based on SERS using silver nanorods, which significantly amplify the signal, has been developed for rapid detection of trace levels of viruses with a high degree of sensitivity and specificity. The technique measures the change in frequency of a near- infrared laser as it scatters viral DNA or RNA. That change in frequency is as distinct as a fingerprint. This novel SERS assay can detect spectral differences between viruses, viral strains, and viruses with gene deletions in biological media. The method provides rapid diagnostics (60 s) for detection and characterization of viruses generating reproducible spectra without viral manipulation. This method is also inexpensive and easily reproducible.

The use of nanoparticles as tags or labels allows for the detection of infectious agents in small sample volumes directly in a very sensitive, specific and rapid format at lower costs than current in-use technologies. This advance in early detection enables accurate and prompt treatment.

Quantum dot technology is currently the most widely employed nanotechnology in this area. The recently emerging cantilever technology is the most promising. The technology strengthens and expands the DNA and protein microarray methods and has applications in genomic analysis, proteomics, and molecular diagnostics.

Waveguide technology is an emergent area with many diagnostic applications. Nanosensors are the new contrivance for detection of bioterrorism agents. All these new technologies would have to be evaluated in clinical settings before their full import is appreciated and accepted.

Nanotechnology in water microbiology – water treatment by detection of microbial pathogens

An adequate supply of safe drinking water is one of the major prerequisites for a healthy life, but waterborne diseases is still a major cause of death in many parts of the world, particularly in young children, the elderly, or those with compromised immune systems. As the epidemiology of waterborne diseases is changing, there is a growing global public health concern about new and reemerging infectious diseases that are occurring through a complex interaction of social, economic, evolutionary, and ecological factors.

An important challenge is therefore the rapid, specific and sensitive detection of waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. However, newer enzymatic, immunological and genetic methods are being developed to replace and/or support classical approaches to microbial detection. Moreover, innovations in nanotechnologies and nanosciences are having a significant impact in biodiagnostics, where a number of nanoparticle-based assays and nanodevices have been introduced for biomolecular detection.

Reprinted from http://www.nanowerk.com/spotlight/spotid=23695.php.

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Scientists Develop Nanomechanical Biosensor Based on Photonic Crystal Nanowire Array

Yuerui Lu, a student of Amit Lal, Cornell University’s Professor of electrical and computer engineering, has developed a photonic crystal nanowire array-based biosensor, which is capable of performing low-cost, highly sensitive and rapid test for detecting disease markers and similar molecules at ultra-low concentrations, paving the way to identify diseases at an early stage.

The nanomechanical biosensor is a 50-µm diameter mechanical resonator made of a thin membrane of silicon-silicon dioxide comprising closely stacked, neatly arranged vertical nanowires over it. This pattern provides a high surface-to-volume ratio for delivering high sensitivity to detect biomolecules at ultra-low concentrations down to fM.

A schematic drawing of the biosensor, which consists of ordered nanowires on top of a silicon-silicon dioxide membrane, may be viewed by clicking on the image at the left (Credit: Yuerui Lu).

The biosensor operates by bonding the nanowires with the single-stranded probe DNA molecules. When the molecules are brought in touch with a target single-stranded DNA, the respective molecules join together, causing a change in the mass recorded by the device, which in turn changes the device’s resonance frequency.

When the device is irradiated by a laser beam, the novel design of the device’s nanowires enables the absorption of over 90% of the light, causing an effective opto-thermo-mechanical agitation of the resonator. The change in the resonance frequency can be optically recorded rapidly and remotely without the use of electrical wires, facilitating the fabrication of the device at a lower cost.

Lal stated that the device can be used for clinical analysis, for instance in DNA testing wherein current methods compare DNA against a typical sequence, which are expensive and time consuming. The novel device can be coded with specific DNA series based on relevance, and those particular molecules can be identified in early stages when at lower concentrations, he said. Doctors can have a cartridge comprising a sequence of membrane sensors that allow the detection of DNA defects rapidly, he added.

The biosensor can be utilized for environmental monitoring purposes such as monitoring of water quality. The research team expects to upgrade the sensitivity of its device to some protein molecules, which is a challenge to the team, as those molecules do not attach as efficiently as DNA molecules do.

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HPLC of PCR Products Now Being Used to Detect Pathogens in Food

Recent developments in rapid methods have led researchers to utilize multiplex polymerase chain reaction (PCR) and high-performance liquid chromatography (HPLC) for the identification of food borne pathogens, such as diarrheagenic E. coli. A recent overview from separationsnow.com discusses this new application, and I have provided an excerpt below.

Dangers of Diarrhoea

Children are at higher risk of contracting diarrhoea than adults due to their underdeveloped immune systems and they are likely to be affected for longer periods. In the Western world, most cases are easily treated but it is a different story in developing countries, where infection rates are higher and death is a common outcome.

Acute diarrhoea is generally caused by bacterial, viral, or parasitic infection and one of the key bacteria is diarrhoeagenic Escherichia coli, which can enter the body via contaminated food. However, it is not simply one strain that is responsible. In foods, four main categories can cause diarrhoea: enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorraghic (EHEC) and enteroinvasive (EIEC) E. coli.

Each of these categories contains similar but individual DNA, which proved a blessing for a team of Chinese scientists who have exploited the differences in a novel detection method. Lichun Cui from the Northeast Forestry University, Harbin, with colleagues from the Northeast Agricultural University, Harbin, and the Heilongjiang and Hainan Entry-Exit Inspection and Quarantine Bureaus, devised a procedure based on denaturing HPLC that could detect single or mixed E. coli infections in food.

Denaturing HPLC

In denaturing HPLC, mismatches in the DNA bases of double-stranded DNA allow their separation provided certain criteria are met. At elevated temperatures, the hetero and homo duplex chains unwind, or denature, to their individual strands which are resolved on the HPLC column.

The HPLC stationary phase must be inert and electrically neutral. Under these conditions, DNA cannot bind due to its inherent negative charge but the addition of an ion pairing agent to the mobile phase changes the properties and binding is achieved via electrostatic interactions.

The hetero duplexes are denatured to a greater extent than the homo duplexes, so that they are retained less strongly on the column and elute first. So, pairs of hetero and homo chains are observed in the HPLC chromatograms.

In the case of E. coli, the researchers employed a poly(styrene-divinylbenzene) column and added triethylammonium acetate to the mobile phase for ion pairing. Separation was effected with a gradient of acetonitrile.

DNA was extracted from E. coli bacteria and subjected to polymerase chain reaction (PCR) amplification using unique primers based on the conserved regions of each of the four bacteria. Sufficient sample was generated for HPLC analysis and only the expected products were produced, as proven by agarose gel electrophoresis. The average size of the products was 220, 300, 330 and 500 base pairs for ETEC, EPEC, EIEC and EHEC, respectively.

Bacterial Strains Detected Together in Food

The HPLC separations were carried out at 50°C under non-denaturing conditions, which produced a single peak for each amplified fragment. They eluted at different retention times over 4-9 minutes, allowing them to be distinguished from each other.

The PCR products from all four E. coli categories were then mixed together for HPLC, confirming that they can be separated and distinguished using their retention times.

The critical step in the process is the specificity towards each category. This was assured by subjecting the genomic DNA from 34 bacterial strains to the same amplification and analysis process using the unique primers. Only the ETEC, EPEC, EIEC and EHEC strains and their isolates gave positive results.

The novel procedure was used to test 189 samples of faecal matter from patients as well as 690 import and export food samples, including beef, pork, chicken, sausages and milk. Blind testing was carried out and the results were compared with those from the conventional method which involves bacterial culture over 2-3 days and biochemical reactions.

A total of 60 positive samples were identified with all four strains being detected. The results were in perfect agreement with the conventional method, confirming the validity of the approach. However, the PCR denaturing HPLC method is much faster and provides a valid alternative. It could also replace the basic PCR assay which requires a gel electrophoresis step.

The multiplex method is capable of detecting all four strains in one procedure, so can identify one or more of the infections in contaminated food and in patient faeces, permitting rapid detection and diagnosis and reducing the time before appropriate action can be taken.

The original article appears here. Image by Renjith Krishnan.

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A Fast Nanotechnology Platform to Detect/Capture Bacteria in Clinical Samples

The folks at Nanowerk (http://www.nanowerk.com/) recently highlighted a novel use of Surface-Enhanced Raman Spectroscopy (SERS) that can be used for label-free sensing of bacteria. A dual function biochip is now being utilized to capture bacteria in blood samples, followed by analysis of the bacteria using Raman spectroscopy. The image on the left (click on the image for a larger picture) illustrates the core of the biochip, where an array of silver nanoparticles (silver) coated by Vancomycin (green) selectively capture bacteria (white) while blood cells (red) are excluded. The following discussion is excerpted from the Nanowerk article, in which Dr. Yuh-Lin Wang, Distinguished Research Fellow, Institute of Atomic and Molecular Sciences, Academia Sinica, and Professor in the Department of Physics at National Taiwan University, describes the technology (Image: Dr. Wang, Academia Sinica. Correspondence and requests for materials should be addressed to Prof. Wang, email: ylwang@pub.iams.sinica.edu.tw).

Surface-enhanced Raman spectroscopy (SERS) is a powerful research tool that is being used to detect and analyze chemicals as well as a non-invasive tool for imaging cells and detecting cancer. It also has been employed for label-free sensing of bacteria, exploiting its tremendous enhancement in the Raman signal.

SERS can provide the vibrational spectrum of the molecules on the cell wall of a single bacterium in a few seconds. Such a spectrum is like the fingerprints of the molecules and therefore could be exploited as a means to quickly identify bacteria without the need of a time-consuming bacteria culture process, which typically takes a few days to several weeks depending on the species of bacteria.

To practically apply SERS to the early diagnosis of bacteremia – the presence of bacteria in the blood – it is most desirable to be able to capture bacteria in a patient's blood onto the SERS substrate.

"A typical SERS-active substrate consists of arrays of nanoscale metallic objects, for example, silver nanoparticles and etch-pits on silver surfaces, which can sustain surface plasmon polariton resonance and enhance the Raman signal of molecules on or near the substrate," Dr. Yuh-Lin Wang, explains. "In our recent work, we found that coating a thin layer of vancomycin on a SERS substrate drastically increases its capability to capture bacteria in the blood samples without introducing significant spectral interference to SERS spectrum of the captured bacteria."

Previously, researchers already used vancomycin-coated magnetic nanoparticles to capture bacteria in water. Wang and his team therefore asked the question whether it is possible to endow the vancomycin-coated SERS substrates with the concurrent functionalities of bacterial capturing and sensing.

Reporting their work in the November 15, 2011 issue of Nature Communications ("Functionalized arrays of Raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood"), first-authored by Ting-Yu Liu, they demonstrate that functionalization by vancomycin of substrates of silver nanoparticles on arrays of anodic aluminum oxide nanochannels not only dramatically enhances their ability to capture bacteria in liquid but also significantly increases their SERS signal.

Wang notes that the team's findings took them by surprise since the signals from the vancomycin coating were expected to be larger than that from the bacteria cell wall since the later is closer to the SERS substrate.

"This unexpected discovery opens up many possibilities for the creation of SERS-based multifunctional biochips for rapid culture-free and label-free detection and drug-resistant testing of microorganisms in clinical samples," he says.

Coating SERS substrates by vancomycin, which is an antibiotic by itself, in order to add the function of capturing bacteria to the substrates is a brand-new approach to overcome a major obstacle facing the practical application of SERS in clinical diagnosis.

"We have been making various attempts to overcome this obstacle in the last two years" says Wang. "This exciting result is the outcome of an experiment that makes some sense superficially but is considered unlikely to work on second thought because of the likely interference from the vancomycin coating. After we saw the surprisingly good result that the SERS signals from the vancomycin coating do not interfere with that of the captured bacteria, we pondered on the question "why" for months and finally came up with some qualitative explanation as given in our paper."

"In retrospect, this is an interesting case in which the original motivation was to tackle the conceptually simple and practically challenging problem of trying to capture bacteria onto a small area of a biosensor without compromising its original sensing function. It turns out that our solution not only allows us to capture bacteria ~1000 times more effectively but also enhances the sensitivity of the sensor by several times rather than decreasing it."

To demonstrate the bacterium-capturing capability of their substrate, the researchers immersed it in a water sample with ultra-low concentration (100 cfu/ml) of bacteria for 1 hour and then rinsed it in deionized water. Examining the substrate with scanning electron microscopy (SEM), they were then able to detect a concentration of bacteria on their substrate.

Wang and his team point out that their findings are a major step towards the development of a high-speed and -sensitivity nanotechnology platform that has high potential to capture/detect bacteria in clinical or environmental samples.

This SERS-based rapid detection method is a very promising approach to dramatically reducing the time needed to detect bacteria in the blood of bacteremia patients to within an hour. By contrast, a conventional biological assay usually requires the sample preparation time ranging from days for fast growing bacteria to weeks for slow growers.

The researchers point out that, in principle, this sensing platform could be exploited for the detection of various microorganisms such as virus and bacteria in various clinical samples, e.g., water, phlegm, sputum, blood and marrow, as well as food, and environmental samples.

"Culture-free and label-free detection of microorganisms remain among the most exciting directions in the development of rapid biosensing technology," says Wang. "One of the most difficult challenges is the development of complementary sample preparation technologies. In order for a new method to be accepted by the research community, scientists and practitioners are looking for a total rather than partial solution."

Reprinted with permission from http://www.nanowerk.com/spotlight/spotid=23548.php.

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NEW RMM Tutorial Pages Launched at rapidmicromethods.com

In connection with the launch of the new RMM Product Matrix, rapidmicromethods.com has developed a series of new scientific tutorial pages. The tutorial pages provide in-depth reviews of the benefits of implementing RMMs, the science behind the technologies, why they differ from conventional microbiology methods, and how they can be applied in the modern microbiology lab and manufacturing environment. Separate pages discuss RMM technologies based on growth, viability staining and laser excitation, the detection of cellular components, optical spectroscopy, nucleic acid amplification and gene sequencing, and Micro-Electrical-Mechanical Systems such as biosensors and microarrays. Visit the new RMM Tutorial pages at http://rapidmicromethods.com/files/tutorial.html.

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NEW Rapid Methods Product Matrix Launched on rapidmicromethods.com

Rapidmicromethods.com has launched an innovative resource for directly comparing more than 45 different rapid method technologies. The RMM Product Matrix provides details on scientific methods, applications, time to result, throughput, sensitivity, organisms detected, identification libraries and product workflow in three separate comparison tables (microbial identification, qualitative and quantitative methods). Never before has this much information been available in one place anywhere online or in print. Users can now use the Product Matrix to assist in matching the right RMM technology with their microbiology applications. Visit the new RMM Product Matrix at http://rapidmicromethods.com/files/matrix.html.

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