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Fast Biosensors for Pathogens in Food Using Sensitive Micro-Cantilevers Array
Reporter: Danut Dragoi, PhD
The novel biosensor developed by scientists at Rice University in collaboration with colleagues in Thailand and Ireland may make the detection of pathogens much faster and easier for food-manufacturing plants, see link in here .
How is it working?
The picture below taken from, see link here, shows an array of sensitive cantilevers that are functionalized with a specific antibody or peptide that binds to a pathogen that we believe is in the food we like to inspect. A general working principle for cantilevers is shown in here, where a bimorph piezoelectric materials, the sensor, with four electrodes deposited in an asymmetric position on the two parallel sides of a cantilever produce an electric signal once the tip is bended or is under a small weight or force. Other micro-cantilevers use different sensors to maximize the sensitivity. If the pathogen binds specifically to a deposited antibody or peptide on a sensitive cantilever, the free tip of the cantilever will deflect down under the weight of the pathogen. The deflection down of the micro-cantilever can be associated with a reflected laser beam deviation on a position sensitive detector that outputs an electrical signal that can be further processed. In this way, we know what specific pathogen is binding to a known micro-cantilever, so that we can identify the pathogen and from the strength of the signal, the amplitude of the deviation, we may be able to say how much or what concentration of the pathogen is in the food. A laser version of the electronic micro-cantilever exists, see link in here. The picture below shows an array of seven micro-cantilever that binds to seven different pathogens due to the fact that each micro-cantilever has different active antibody or peptide specific to bind on a pathogen.
Figure below shows construction details of one cantilever used by the authors of the paper published, link in here. The yellow color designates the Gold layer deposited on a Si substrate. This Au/Si micro-cantilever shown do not use a sensor attached to it because it may cause a poor sensitivity to small amounts of pathogens detected.
A study on this research appears online this month in the American Chemical Society journal Analytical Chemistry, see link in here.
Advantages of the technique
The process appears to easily outperform tests that are now standard in the food industry. The standard tests are slow because it can take days to culture colonies of salmonella bacteria as proof, or laborious because of the need to prepare samples for DNA-based testing.
The Rice process delivers results within minutes from a platform that can be cleaned and reused. The technology can be easily customized to detect any type of bacteria and to detect different strains of the same bacterium.
The “diving boards” are a set of microcantilevers, each of which can be decorated with different peptides that have unique binding affinities to strains of the salmonella bacteria. When a peptide catches a bacterium, the cantilever bends over so slightly, due to a mismatch in surface stress on the top and bottom. A fine laser trained on the mechanism catches that motion and triggers the alarm.
The system is sensitive enough to warn of the presence of a single pathogen, according to the researchers, who wrote that very low pathogen concentrations cause foodborne disease.
The authors
From the article published on line, see link in here, we can envision the direction and the applications of the research in the future.
The idea springs from research into the use of microcantilevers by Rice biomolecular engineer Sibani Lisa Biswal and lead author Jinghui Wang, a graduate student in her lab. Biswal was prompted to have a look at novel peptides by her graduate school friend, Nitsara Karoonuthaisiri, head of the microarray laboratory at the National Center for Genetic Engineering and Biotechnology in Thailand. Karoonuthaisiri is also a visiting scientist at the Institute for Global Food Security at the Queen’s University, Belfast.
“She’s been working in this area of pathogenic bacteria and asked if we have thought about trying to use our microcantilevers for detection,” Biswal said. “Specifically, she wanted to know if we could try these novel peptides.”
Karoonuthaisiri and her team had isolated bacteriophage viruses associated with salmonella through biopanning and phage display, a technique to study interactions among proteins, peptides and pathogens. She then derived peptides from the phages that would serve as targets for specific bacteria.
“She said, ‘We spend a lot of time trying to characterize which of these peptides work the best. It looks like you have a platform that can do and quantitate that.’ So that’s where we came in,” Biswal said.
The Rice lab compared the peptides’ performance with commercial antibodies now used for salmonella detection and found the peptides were not only more sensitive but could be used in a multiplexed cantilever array to detect many different kinds of salmonella at once.
“The peptides are very robust,” Biswal said. “That’s why a lot of people like them over antibodies. The peptides can handle harsher conditions and are much more stable. Antibodies are large proteins and break down more readily.
“We’re very excited to see where this will lead,” she said.
Our comment
If the peptide adherence on the cantilever is strong, as the authors suggest, then different microcantilever made of Quartz can be used, knowing that extremely small amounts of pathogen bonded will change the frequency of the microcantilever.In this case the calibration curve is frequency versus weight.
Jinghui Wang†, M. Josephine Morton‡, Christopher T. Elliott‡, Nitsara Karoonuthaisiri‡§, Laura Segatori†, and Sibani Lisa Biswal*†, Rapid Detection of Pathogenic Bacteria and Screening of Phage-Derived Peptides Using Microcantilevers, Anal. Chem., 2014, 86 (3), pp 1671–1678
Publication: Rapid Detection of Pathogenic Bacteria and Screening of Phage-Derived Peptides Using Microcantilevers. Jinghui Wang, M. Josephine Morton, Christopher T. Elliott, Nitsara Karoonuthaisiri, Laura Segatori, and Sibani Lisa Biswal. Analytical Chemistry (Article ASAP): http://pubs.acs.org/doi/abs/10.1021/ac403437x
Combining Nanotube Technology and Genetically Engineered Antibodies to Detect Prostate Cancer Biomarkers
Writer, Curator: Stephen J. Williams, Ph.D.
Article ID #61: Combining Nanotube Technology and Genetically Engineered Antibodies to Detect Prostate Cancer Biomarkers. Published on 6/13/2013
WordCloud Image Produced by Adam Tubman
Article 9.4.Combining Nanotube Technology and Genetically Engineered Antibodies to Detect Prostate Cancer Biomarkers
Figure of Carbon Nanotube Transistor design with functionalized antibodies for biomarker detection. From paper of A.T. Johnson; used with permission from A.T. Johnson)
In a literature review of the current status of the breast cancer biomarker field[2], author Dr. Michael Duffy, from University College Dublin, pondered the clinical utility of breast cancer serum markers and suggested that due to lack of sensitivity and specificity none of available markers is of value for detection of early breast cancer however these biomarkers have been shown useful in monitoring patients with advanced disease. For instance high preoperative CA15-3 is indicative of adverse patient outcome. According to American Society of Clinical Oncology Expert Panel, however CA 15-3 may lack the sensitivity and disease specificity for breast cancer as a prognostic marker. For panel suggestions please click on the link below:
The same panel also concurred on the lack of prognostic value of other markers (for example CEA for colon cancer) but did agree that 66-73% of patients with advanced disease, who responded to therapy, showed reduction in these serum markers. Indeed, CA125, long associated as a biomarker for ovarian cancer, does not have the sensitivity and especially the disease specificity to be a stand-alone prognostic marker[3]. Therefore, although “omics” strategies have suggested multiple possible biomarkers for various cancers, a major issue in translating a putative biomarker to either:
1) a clinically validated (panel) of disease-relevant biomarkers or
2) biomarkers useful for therapeutic monitoring
is obtaining the specificity and sensitivity for detection in bio-specimens. As discussed below, this is being achieved with the merger of nanotechnology-based sensors and bioengineering of biomolecule.
For ASCO panel suggestions of biomarkers useful in Prostate cancer please see the link below:
Osteopontin is a phosphorylated glycoprotein secreted by activated macrophages, leukocytes, activated T lymphocytes and is present at sites of inflammation (for a review of OPN see [4]). Osteopontin interacts with several integrins and CD44 (a putative cancer stem cell marker). Binding of OPN to cell integrins mediates cell-matrix and cell-cell communication, stimulating adhesion, migration (through interaction with urokinase plasminogen activator {uPA}) and cell signaling pathways such as the HGF-Met pathway. Overexpression is found on a variety of cancers including breast, lung, colorectal, ovarian and melanoma[5]. And although OPN is detected in normal tissue, it is known that OPN over-expression can alter the malignant potential of tumor cells.
Roles of osteopontin in cancer include:
Binding to CD44
Increase in growth factor signaling (HGF/Met pathway)
Increase uPA activity- increase invasiveness
Angiogenesis thru binding with αvβ3 integrin and increased VEGF expression
Protection against apoptosis: OPN activates nuclear factor Κβ
Some researchers have suggested it could be a prognostic marker for breast and lung cancer while there have been conflicting reports as to whether OPN expression is correlated to malignant potential in prostate cancer[6]. Osteopontin is found on tumor infiltrating macrophages, which may contribute to OPN as a prognostic marker. Breast cancer patients (disseminated carcinomas) have 4-10 times higher serum levels of OPN than found in healthy patients, although there is no difference in pre- or post-menopausal women[7].
Piezoelectric sensors have been used by the same group at Fox Chase Cancer Center to detect serum levels of the HER2 protein in breast cancer patients, for the purpose of therapeutic monitoring after anti-HER2 antibody trastuzumab (Herceptin™) therapy. Lina Loo, in the laboratory of Dr. Gregory Adams showed the utility of using (scFv) to trastuzumab (anti-HER2) with pizo-electric nanotubes to accurately and reproducibly determine levels of serum HER2[8]. This method improved the sensitivity of serum HER2 detection over other methods such as:
However the advent of nanotechnology-based detection system combined with engineered affinity-based biomolecules has increased both the sensitivity and specificity of biomarker detection from complex fluids such as plasma and urine. The advent of multiple types of biosensors, including
has given the ability to measure, with enhanced sensitivity and specificity, putative biomarkers of disease in minute volumes of precious bio-samples.
The basic design of a biosensor is made of three components:
A recognition element (I.e. antibodies, nucleic acids, enzymes)
A signal transducer (electrochemical, optical, piezoelectric)
Signal processor (relays and displays)
In the journal ACS Nano Mitchel Lerner from Dr. Charlie Johnson’s laboratory at University of Pennsylvania in collaboration with Fox Chase Cancer researchers in the laboratory of Dr. Matthew Robinson, describe a piezoelectric detection system for quantifying levels of osteopontin (OPN), a putative biomarker for prostate cancer[1]. In this paper Dr. Robinson’s group at Fox Chase, genetically engineered a single chain variable fragment (scFv) protein {the binding portion of the antibody} which had high affinity for OPN. This scFv was attached to a carbon nanotube field-effect transistor (NT-FET), designed by Dr. Johnson’s group, using a chemical process called chemical functionalization {a process using diazonium salts to covalently attach scFV to NT-FET.
Figure. Functionalization scheme for OPN attachment to carbon nanotubes. As figure 1 legend in paper states: “First, sp8 hybridized-sites are created o the nanotube sidewall by incubation in a diazonium salt solution. The carboxylic acid group is then activated by EDC and stabilized with NHS.ScFv antibody displaces the NHS and forms an amide bond. OPN epitope is shown in yellow and the C and N-terminuses are in orange and green respectively.” (used by permision for A.T. Charlie Johnson)
This system was then used to determine the selectivity and sensitivity of OPN from complex solutions.
Methods:
Nanotube (NT) design
Grown by catalytic vapor deposition
Electrical contacts patterned using photo-lithography
Atomic Force microscopy was used to verify structure of nanotube
Chemical Linking of scFv to nanotube
Diazonium treatment resulted in activation and subsequent stabilization of amino (NHS) side chain
Amine group on lysine of scFV displaced NHS group => covalent attachment of scFV to NT
Atomic Force Spectroscopy used to verify linkage of scFv to nanotube
Results showed there was
minimal non-specific binding of OPN to the scFv
system allowed for detection limit of 1 pg/ml OPN (pictogram/milliliter) or 30 fM (fentomolar) in a phosphate buffered saline solution.
Only a minute volume (10 µl) of sample is needed
Sensor able to measure million-fold range of OPN concentrations ( from 10-3 to 103 ng/mL OPN)
Two experiments were conducted to determine the specificity of OPN to the antibody-detection system.
1st experiment
– scFv functionalized sensor was incubated in a solution of high concentration of BSA (450 mg/ml) to approximate nonspecific proteins in patient samples
– minimal signal was detected
2nd experiment
– Functionalized NT-FET devices with a scFv based on the HER2 therapeutic antibody trastuzumab
– There was no binding of OPN to anti-HER2 devices
– Therefore anti OPN (23C3) scFv-functionalized carbon nanotube sensors exhibit high levels of specificity to OPN
The authors conclude “the functionalization procedure described here is expected to be generalizable to any antibody containing an accessible amine group, and to result in biosensors appropriate for detection of corresponding complementary proteins at fM concentrations”.
I had the opportunity to speak with co-author Dr. Matthew Robinson, Assistant Professor in the Developmental Therapeutics Program at Fox Chase Cancer Center about the next steps for this work. Dr. Robinson mentioned that “at this point we have not looked in patient samples yet but our plan is to move in that direction. We need to establish sensitivity/specificity in increasingly complex samples (e.g. spiked normal serum and retrospectively in patient serum with known levels of biomarkers).”
Cancer patients often present a complex metabolic profile. The paper notes that OPN has a pI (isoelectric point) of 4.2, which would result in a negative charge at physiologically normal pH of 7.6. I asked Dr. Robinson about if changes in metabolic profile could hinder OPN binding to the NT-FET system would require some preprocessing of blood samples. Dr. Robinson agreed “that confounding variables such as additional diseases but even things like diet (i.e. is fasting necessary) need to be addressed before this platform is ready for use in clinical setting. It is likely that sample prep will be needed to remove albumin, lower salt concentrations, etc. This could end up being problematic for biomarkers that are unstable and would degrade over the time necessary for sample prep. It is also possible that sample prep to remove albumin and other background factors could result in loss of biomarkers. This will need to be determined on a case-by-case basis with validated testing methods.” One useful advantage of this system is the possibility of measuring multiple biomarkers, clinically important as studies has suggested that
multiple markers result in the higher sensitivity/specificity for many infrequent cancers, such as ovarian. Dr. Robinson agrees “that panels of biomarkers are likely to be better at early detection and diagnosis. In principle the platform that we describe can be set up to allow for detection of multiple biomarkers at a time. From the biology end of things we have built antibodies against 3 different prostate cancer biomarkers for that purpose.”
Dr. Johnson commented on the ability of the platform allowed for the simultaneous detection of multiple biomarkers, noting that ”the platform is compatible with the measurement of multiple biomarkers through the use of multiple devices, each functionalized with their own antibody.”
ASCO guidelines Expert Panel on Tumor Biomarkers 2007 Updatefor Breast Cancer:
Published in JCO, Vol. 25, Issue 12 (April 20), 2007: 1596-1605
References:
1. Lerner MB, D’Souza J, Pazina T, Dailey J, Goldsmith BR, Robinson MK, Johnson AT: Hybrids of a genetically engineered antibody and a carbon nanotube transistor for detection of prostate cancer biomarkers. ACS nano 2012, 6(6):5143-5149.
5. Brown LF, Berse B, Van de Water L, Papadopoulos-Sergiou A, Perruzzi CA, Manseau EJ, Dvorak HF, Senger DR: Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces. Molecular biology of the cell 1992, 3(10):1169-1180.
6. Thoms JW, Dal Pra A, Anborgh PH, Christensen E, Fleshner N, Menard C, Chadwick K, Milosevic M, Catton C, Pintilie M et al: Plasma osteopontin as a biomarker of prostate cancer aggression: relationship to risk category and treatment response.British journal of cancer 2012, 107(5):840-846.
7. Brown LF, Papadopoulos-Sergiou A, Berse B, Manseau EJ, Tognazzi K, Perruzzi CA, Dvorak HF, Senger DR: Osteopontin expression and distribution in human carcinomas. The American journal of pathology 1994, 145(3):610-623.
8. Loo L, Capobianco JA, Wu W, Gao X, Shih WY, Shih WH, Pourrezaei K, Robinson MK, Adams GP: Highly sensitive detection of HER2 extracellular domain in the serum of breast cancer patients by piezoelectric microcantilevers. Analytical chemistry 2011, 83(9):3392-3397.
Other posts from this site on Biomarkers, Cancer, and Nanotechnology include:
The field of DNA and RNA nanotechnologies are considered one of the most dynamic research areas in the field of drug delivery in molecular medicine. Both DNA and RNA have a wide aspect of medical application including: drug deliveries, for genetic immunization, for metabolite and nucleic acid detection, gene regulation, siRNA delivery for cancer treatment (I), and even analytical and therapeutic applications.
Seeman (6,7) pioneered the concept 30 years ago of using DNA as a material for creating nanostructures; this has led to an explosion of knowledge in the now well-established field of DNA nanotechnology. The unique properties in terms of free energy, folding, noncanonical base-pairing, base-stacking, in vivo transcription and processing that distinguish RNA from DNA provides sufficient rationale to regard RNA nanotechnology as its own technological discipline. Herein, we will discuss the advantages of DNA nanotechnology and it’s use in medicine.
So What is the rational of using DNA nanotechnology(3)?
Genetic studies – its application in various biological fields like biomedicine, cancer research, medical devices and genetic engineering.
Its unique properties of structural stability, programmability of sequences, and predictable self-assembly.
Structures made from DNA using the DNA-origami method (Rothemund, 2006)
Structural DNA nanotechnology rests on three pillars: [1] Hybridization; [2] Stably branched DNA; and [3] Convenient synthesis of designed sequences.
Hybridization
Hybridization. The self-association (self=assembly) of complementary nucleic acid molecules or parts of molecules, is implicit in all aspects of structural DNA nanotechnology. Individual motifs are formed by the hybridization of strands designed to produce particular topological species. A key aspect of hybridization is the use of sticky ended cohesion to combine pieces of linear duplex DNA; this has been a fundamental component of genetic engineering for over 35 years (7). Not only is hybridization critical to the formation of structure, but it is deeply involved in almost all the sequence-dependent nanomechanical devices that have been constructed, and it is central to many attempts to build structural motifs in a sequential fashion (7,8 ).
Stably Branched DNA
branched DNA molecules are central to DNA nanotechnology. It is the combination of in vitro hybridization and synthetic branched DNA that leads to the ability to use DNA as a construction material. Such branched DNA is thought to be intermediates in genetic recombination (such as Holliday junctions).
Convenient Synthesis of Designed Sequences
Biologically derived branched DNA molecules, such as Holliday junctions, are inherently unstable, because they exhibit sequence symmetry; i.e., the four strands actually consist of two pairs of strands with the same sequence. This symmetry enables an isomerization known as branch migration that allows the branch point to relocate. DNA nanotechnology entailed sequence design that attempted to minimize sequence symmetry in every way possible.
One of the most remarkable innovations in structural DNA-nanotechnology in recent years is DNA origami, which was invented in 2006 by Paul Rothemund (1) (see Fig above). DNA origami utilizes the genome from a virus together with a large number of shorter DNA strands to enable the creation of numerous DNA-based structures (Figure 1). The shorter DNA strands forces the long viral DNA to fold into a pattern that is defined by the interaction between the long and the short DNA strands (1,2).
Rothemund believes that an application of patterned DNA origami would be the creation of a ‘nanobreadboard’, to which diverse components could be added. The attachment of proteins23, for example, might allow novel biological experiments aimed at modelling complex protein assemblies and examining the effects of spatial organization, whereas molecular electronic or plasmonic circuits might be created by attaching nanowires, carbon nanotubes or gold nanoparticles (1).
DNA nanotechnology and Biological Application
The physical and chemical properties of nanomaterials such as polymers, semiconductors, and metals present diverse advantages for various in vivo applications (3,9 ). For example:
Therapeutics – In cancer for example, nanosystems that are designed from biological materials such as DNA and RNA are ‘programmed’ to be able to evade most, if not all, drug-resistance mechanisms. Based on these properties, most nanosystems are able to deliver high concentrations of drugs to cancer cells while curtailing damage to surrounding healthy cells (2b, 3, 9, 11, 15).
Biosensors – capable of picking up very specific biological signals and converting them into electrical outputs that can be analyzed for identification. Biosensors are efficient as they have a high ratio of surface area to volume as well as adjustable electronic, magnetic, optical, and biological properties (3, 12, 13, 14).
**Amin and colleagues have developed a biotinylated DNA thin film-coated fiber optic reflectance biosensor for the detection of streptavidin aerosols. DNA thin films were prepared by dropping DNA samples into a polymer optical fiber which responded quickly to the specific biomolecules in the atmosphere. This approach of coating optical fibers with DNA nanostructures could be very useful in the future for detecting atmospheric bio-aerosols with high sensitivity and specificity (3, 14)
Computing – Another aspect uses the programmability of DNA to create devices that are capable of computing. Here, the structure of the assembled DNA is not of primary interest. Instead, control of the DNA sequence is used in the creation of computational algorithms, like e.g. artificial neural networks. Qian et al for example, built on the richness of DNA computing and strand displacement circuitry, they showed how molecular systems can exhibit autonomous brain-like behaviours. Using a simple DNA gate architecture that allows experimental scale-up of multilayer digital circuits, they systematically transform arbitrary linear threshold circuits (an artificial neural network model) into DNA strand displacement cascades that function as small neural networks (3, 10).
Additional features: 3rd generation DNA sequencers (II), Biomimetic systems, Energy transfer and photonics etc
Summary:
DNA nanotechnology is an evolving field that affects medicine, computation, material sciences, and physics. DNA nanostructures offer unprecedented control over shape, size, mechanical flexibility and anisotropic surface modification. Clearly, proper control over these aspects can increase circulation times by orders of magnitude, as can be seen for longcirculating particles such as erythrocytes and various pathogenic particles evolved to overcome this issue. The use of DNA in DNA/protein-based matrices makes these structures inherently amenable to structural tunability. More research in this direction will certainly be developed, making DNA a promising biomaterial in tissue engineering. future development of novel ways in which DNA would be utilized to have a much more comprehensive role in biological computation and data storage is envisaged.
3. Muniza Zahid, Byeonghoon Kim, Rafaqat Hussain, Rashid Amin and Sung H Park. DNA nanotechnology: a future perspective. Nanoscale Research Letters 2013, 8:119. http://www.nanoscalereslett.com/content/8/1/119
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