Category Archives: Biosensor

Biosensors using Magnetic NanoTags (MNTs)

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.

Label Free Biosensors: A List

Since the release of first label free biosensor -Biacore – in early 1990s the label free biosensors have become gold standards for measuring biomolecular interactions. Before the introduction of label free biosensing technology the predominant means for detection biomolecular interaction was based on ELISA/FIA format. Though widely used, these methods required binding partners to be labeled with enzyme, fluorophore or radiolabel for detection. Labels invariably affected the binding kinetics of interaction by modifying the structure of binding partners. In addition, the measurements were typically end point assay making it difficult to get association and dissociation constants of the binding reaction. Introduction of Biacore fundamentally altered the way the biomolecular interactions are studied by enabling measurement of binding event in real time. Since the introduction of Biacore, several other label free biosensing platforms have been introduced in the market. Most of them are based on optical properties like surface Plasmon resonance, diffraction gratings and Total internal Reflection. However, new technologies are being introduced into the market on a regular basis. For example measuring heat released or absorbed during binding event to measure kinetics or using micro-cantilever technology based on Atomic Force Microscopy platform to measure change in height. Below you will find a list of companies offering Label Free Biosensors.

Please let me know if I have missed some companies. Please acknowledge the site if you are disseminating this information anywhere else.

The list of companies has been updated  with clickable links  and is available in Resources page (Dec 28; 2009): Updated List of Label Free Biosensing Companies 

NanoSensors: Nanoparticles as Chemo/Bio Sensors

 

Nanotechnology offers new options for design of chemical sensors and biosensors due to their unique features including

  • Tunable shape and size dependent chemical and physical properties
  • Miniaturization and low power requirements for portable and implantable devices
  • Multiplexing: Several nanomaterials (e.g. nanoparticles, nanorods, nanowires, core-shell nanospheres) each with unique sensitivity to a specific analyte can be integrated on a single chip like platform
  • Nanosensors could be integrated into clothes for wearable, real-time personal sensing devices for environmental monitoring and health monitoring
  • Nanomaterials can be used from designing inexpensive portable environmental sensor to single molecule detection inside the cell.

Rapid use of nanomaterials in design of novel biosensor is evident from three recent reports of designing novel sensors for applications as varied as diagnostics, environmental monitoring, and for sensing inside a cell.

Gold Nanoparticles for Detection of Lung Cancer

Most common applications of Gold Nanoparticles for sensing leverage changes in optical properties in response to some binding event. But Hossam Haick at Haifa, Israel uses conducting properties of Gold nanoparticle coated gold film for sensing varieties of organic molecules present in the breath to diagnose Lung Cancer.

Schematic of Chemoresistive sensor based on Gold nanoparticles ().

First, around 300-400 volatile organic compounds (VOCs) were identified in the breath samples using GC-MS (Gas chromatography-Mass spectroscopy). That number was narrowed down to 33 VOCs that were present at different concentrations and in different composition in normal healthy volunteers and Lung cancer patients. Second, nine chemoresistive sensors were fabricated each containing 5nm gold nanoparticles decorated with monolayer of organic thiol molecules. Each sensor responds differently to the presence of panel of 33 VOCs in the breath of the person. The panel of nine sensors was able to diagnose Lung cancer patients simply by analyzing the breath samples.

This sensor architecture can be easily mass produced and has the potential for use in any number of application.

 

Europium Doped Silica Nanoparticles for Detection of Reactive Oxygen Species (ROS) Inside Cells

The principle behind this sensor , developed by group at CNRS, France, is that silica nanoparticles doped with europium undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants (e.g. H2O2 etc.), leading to recovery of luminescence. The sensors can detect H2O2 inside a cell and has dynamic range of 1-45micromolar. The sensor can also be regenerated and are capable of time-resolved detection of ROS. These nanoparticles are not toxic to the cells and may be targeted to various cell compartment by appropriate functionalization.

 

Single Walled Carbon Nanotubes (SWNTs) Sensor for Oxygen Detection

This sensor proposed by the group at University of Pittsburg combines the best of above two sensors. First, a chemoresistive sensor is made by SWNT networks decorated with an oxygen-sensitive Europium containing dendrimer. Second, when this sensor is illuminated with 365 nm light, it shows optical spectroscopic and electrical conductance sensitivity towards oxygen gas at room temperature under ambient pressure. The sensor has linear sensitivity towards oxygen gas in the environmentally relevant concentration range of 5–27%.

 

These three reports are just a snapshot of large arrays of sensing platforms being designed using nanomaterials. Hopefully, we will soon see a transition of these technologies from research laboratories to the commercial products. Just a reminder though health risk of nanoparticles is of growing concern and will have to be taken into concern before disposable/wearable/bedside nanosensors become a reality

Mix and Match Gold Nanoparticles with GFPs for Serum Protein Biosensor (Chemical Nose)

 

Plasma (the solution fraction of blood after removal of blood cells) or serum (the solution fraction of blood after removal of blood cells and clotting factors) is perhaps the most complex protein mixture containing

  • May be greater than 100,000 proteins and proteins variants if post translation modifications (PTMs), truncation, splice variants, degradation products, precursor and mature proteins and finally all the immunoglobulin variants are taken into account
  • Protein concentration range of 10 orders of magnitude: Albumin is present at 50mg/ml whereas interleukins are present in sub pg/ml range

Change in abundance of plasma/serum protein level and/or protein structural changes are responsible for majority if not all of the human diseases. Considering the importance of plasma/serum proteome (collection of all the proteins in plasma/serum) a simple, inexpensive and easy to implement detection method for quantitation of proteins in plasma/serum will be very attractive for diagnostics. Prof. Rotello and his group have come up with a proof-of-principle concept for serum protein sensor by combining gold nanoparticles and Green fluorescent proteins (GFPs). Concept is simple and elegant: when gold nanoparticles (NPs) decorated with positively charged ligands are added to the GFP proteins solution (pI=5.92 hence negatively charged at physiological pH) the fluorescence of GFPs is quenched due to the formation of NP-GFP complex (because of electrostatic attraction).

Credit www.nature.com/naturechemistry

When other proteins are added to NP-GFP complex the GFP is either released from NP-GFP complex and result in increase in fluorescence or added proteins complex with GFPs further reducing the fluorescence. As it turns out the increase and decrease in fluorescence is protein dependent as well as dependent on the ligands decorating gold NPs. When specific proteins are mixed with a panel of five gold NPs decorated with different ligands each increase and decrease of fluorescence create a pattern/signature that is specific for each protein. Using this approach, researchers demonstrated that they could detect five most abundant serum proteins in buffer as well as in serum. A similar trick was used by them to create a ‘chemical nose’ to sniff out cancer cells.

This technology has the potential to be implemented as a bed side diagnostic device but several challenges remains especially

  1. Detection of low abundant proteins. Cytokines in serum are important indicators of inflammations among other things and are typically present in pg/mL range
  2. Can the sensor distinguish between native proteins or proteins modified by PTMs, truncation, and degradation?
  3. Cost and multiplexing capabilities

Will keep an eye on next generation of “chemical noses”!

Multiplexed Blood Test for Prognosis of Ovarian Cancer

Vermillion (www.vermillion.com) and Quest Diagnostics (www.questdiagnostics.com) got FDA approval for their OVA1TM Test, the first blood test that that can indicate the likelihood of ovarian cancer with high sensitivity prior to biopsy or exploratory surgery. The test is a multiplexed assay simultaneously testing for five ovarian cancer biomarkers−Transthyretin (TT or prealbumin), Apolipoprotein A-1 (Apo A-1), Beta2-Microglobulin (Beta2M), Transferrin (Tfr) and Cancer Antigen 125 (CA 125 II) and then using an algorithm to look for specific pattern to come up with a prognosis. The test in conjunction with other clinical tests will help physician and patients make right choices about the possible treatment and help in reducing mortality rate.

Ovarian cancer is 8th most common cancer among women excluding skin cancer. According to FDA, in 2009 approximately 21,550 new cases of ovarian cancer will be detected and 14,600 deaths will reported as a result of ovarian cancer. Early detection of ovarian cancer is critical because ovarian cancer has a 5-year survival rate of 93 percent when detected early the 5-year survival rate falls to 18 percent for late stage disease.

I have written extensively in my Blog about several multiplexed Assays/biosensing devices under development all targeted toward improving diagnosis or prognosis of diseases and it is nice to see something moving out of Research Lab to a Clinical Laboratory.

In the end it is all about saving lives.

NanoPen for Patterning Nanoparticles

Frankly speaking, my first thought was that this paper is about a nanosized pen to arrange nanoparticles at the surface but when I read the whole things the phrases like “Don’t Judge a book by its cover” or “looks can be deceptive” starts popping up in my mind. The article is not about a tiny tiny pen but rather is about way of arranging nanoparticles in any patter.

Jokes aside, the work by Prof. Wu at Berkley is serious business and presents an easy way to pattern nanoparticles/ nanopatterns over large area not easily achieved by existing patterning tools like soft lithography, dip pen nanolithography or e-beam lithography. According to authors Nanopen is

“— a novel technique for low optical power intensity, flexible, real-time reconfigurable, and large-scale light-actuated patterning of single or multiple nanoparticles, such as metallic spherical nanocrystals, and one-dimensional nanostructures, such as carbon nanotubes”

By exploiting electrokinetic forces, Nanopen is capable of arranging nanoparticles over large surface area (thousands of square micrometers) within few seconds simply by using low power laser light, LEDs or just plain old halogen lamps. The power of Nanopen is shown by arranging 90nm gold nanoparticles in various shapes and sizes including logo of NIH, and a 10×10 array with each individual spots of 10-20µm in size. By placing gold-nanoparticles in close proximity with exact spatial control using NanoPen, authors were able to create ‘hot-spots’ for very sensitive detection of Rhodamine 6G using SERS (Surface Enhanced Raman Spectroscopy). I hope other types of sensors will follow soon.

Gold nanoparticles are extensively used for sensing applications and NanoPen technology opens up new avenues for exploiting these wonderful particles for designing novel biosensors. As noted by authors it is possible to integrate microfluidics channels with NanoPen writing platform to deliver gols nanoparticles activated with different ligands, proteins, DNAs or anything else to design multiplexed detection devices. Gold nanoparticles can also be decorated by various chemical reactive groups using self assembled monolayers (SAMs) of alkanethiols hence it is entirely possible to design a pattern using NanoPen and then use it as template for bottom-up fabrication.

I am no expert in microfabrication but looking at the device design it seems to me that multiple devices can be fabricated in chip format for simultaneous writing of multiple particle types at the same time.

Possibilities are immense but the question is can the technology translate out of lab to a real commercial instrument!

Self Assembled Monolayers (SAMs) on Gold: Celebrating 25 (or so) years!

Recently, I ended up chasing original reference for self assembled monolayers (SAMs) on gold surface and realized that it was 1989 when Nuzzo and Allara first showed the formation of oriented monolayers on gold using dialkyl disulfides. That’s 25 years or so of SAMs and the way this technology is being used (in biology) this may just be the beginning. George Whitesides has been the early driving force in using SAMs for biological applications. A quick search for SAMs on ACS (American Chemical Society) web site (www.pubs.acs.org) throws up more than 10,000 references-Not bad!

My first exposure to SAMs came around 9 years back while designing biointerfaces for protein arrays. Since then I have used this wonderful technology from designing nanoparticle surface to using them for bio-molecular interaction studies. I didn’t find any celebratory article for 25years of SAMs so as a way of my honoring this wonderful technology I decided to catalogue the application of SAMs in biology. I am certain that I will miss several things given the widespread use of SAMs but something is better than nothing.

  1. Biomaterial Research: U.S. Market for biomaterials in 2000 was 9 billion dollars that show the need for such materials for improving quality of life as well as the commercial importance of Biomaterial research. Perhaps the most significant impact of SAMs in Biomedical research has been in design of model surfaces with specific properties for their interaction with biological system. Surfaces that resist protein binding (non-fouling surfaces) or surfaces that can be tailored to interact with biological system in a specific fashion are important to ensure the success of medical implants. SAMs with poly/oligo-ethylene glycol surfaces have been used to design non-fouling surfaces and to demonstrate that degree of polymerization and polymer density determines the non-fouling properties of the surfaces. SAMs have been used to engineer cell position, shape, function in an effort to model cell cultures to mimic real systems. Stem cell differentiation can be controlled by designing surface cues (chemical, topographical, physical) using SAMs. SAMs will continue to play important role in this area.

     

  2. Micro-Contact Printing/Soft-Lithography: Soft lithography uses elastomeric stamp typically made of PDMS (Polydimethyl siloxane) to transfer small patterns (micrometer sized) onto substrates. Patterns of SAMs on Gold allow cells to be placed at specific place and shape to control their function. Proteins can be printed using PDMS for making protein arrays. Numerous other applications uses this trick to pattern proteins, DNAs, peptides, aptamers, cells-you name it and it can be printed! Flexible/Plastic circuits uses SAMs for making contacts

     

  3. Biosensors: SAMs or similar chemistry in gold surfaces is the surface of choice for SPR (surface Plasmon resonance) and QCM (quartz crystal microbalance) biosensors. SAMS are also used for biosensors using microcantilever transducer (see my previous posting). SAMs containing PEGs with reactive end groups are commonly used for specific binding of proteins but at the same time eliminating non-specific binding. Because SAMs enable exquisite control over the density of reactive groups at the surface, the studies on biomolecular interaction as a function of ligand densities have provided useful insight into biology.

     

  4. Bottom-up fabrication: Self assembled monolayers nanopatterns with reactive end groups are used to grow polymers from the surface.

     

  5. Dip-Pen Nanolithorgraphy (DPN): Dip pen nanolithography pioneered by Chad Mirkin uses alkanethiol as ink and AFM (Atomic Force Microscopy) cantilever as pen to write on “Gold Coated Paper”. Since first demonstration a wide variety of inks, including small organic molecules, polymers, DNA, proteins, peptides, colloidal nanoparticles, have been patterned on variety of substrates including insulating, semiconducting and metallic substrates. The most visible use of this technology has been Nano-Art. The picture below was created using DPN. Given this is a serious technology I still can’t resist imagining DPN machines replacing “Name on Rice Grain” machines at State Fairs around the country!

     

    Credit: The International Institute for Nanotechnology. The actual size of the image is smaller than a blood cell.

     

  6. Nanoparticles (Gold/silver) synthesis and chemical functionalization:

     

    1. Monolayer protected clusters (MPC). Reduction of HAuCl4 salt in presence of alkanethiol result in gold nanoparticles of 1-3nm. MPC unlike other gold nanoparticles can be repeatedly isolated from and redissolved in common organic solvents without irreversible aggregation or decomposition. The protective MPC can be made reactive by Ligand Exchange Reaction where an alkanethiol with a reactive group replaces the protective alkanethiol from around the gold nanoparticle
    2. Gold and silver nanostructures and nanoparticles are functionalized using self assembled monolayer’s and used for biosensing. See my previous posting

 

Send me additional information that I am certain I have missed!

Glucose Biosensors and Need for Accuracy

In a previous post I talked about the need for reproducibility and accuracy in multiplexed bioassays but report in today’s NY Times made clear the enormity of the problem even when a single analyte is measured. The key points from the report “Standards Might Rise on Monitors for Diabetics” are

  • In United States 18 million peoples have Diabetes and another 6 million are expected to have it without knowing
  • More than 11 million diabetic in Unites States use Glucose monitors
  • Tight control of blood glucose level resulted in 76, 54 and 60% reduction in the risk for retinopathy, albuminuria and clinical neuropathy respectively
  • International Standard Organization (ISO) allows 20% variation in strip based blood glucose monitor when glucose values are >75mg/dl and values must be within 15mg/dl when Glucose values are less than 75mg/dl. ISO Document#15197 “In vitro diagnostic test systems — Requirements for blood-glucose monitoring systems for self-testing in managing diabetes mellitus”
  • FDA analysis has however showed that stricter standards of 15% variation can be met by most manufacturer
  • Strip based glucose monitors can give erroneous results under various conditions including
    • In presence of Tylenol, vitamin C or sugar such as Xylose or mannose
    • At high temperature, humidity, altitude
    • When blood is taken from alternate site like thigh or forearm
    • Mishandling of strip or instrument
  • A study by government researchers found that when comparing tests from five different popular monitors, results varied by as much as 32 percent
  • A stricter requirement on quality control will increase the price of the testing and may discourage regular testing and hence increase the risk.
  • FDA however agrees that testing of Glucose as biomarkers for diagnosis in ICUs and hospital environment should be done by non-strip based systems with accuracy approaching laboratory based systems
  • Continuous training and awareness is required for proper use of glucose monitoring devices

 

Though FDA/ISO guidelines and specific need for meeting quality standards may be common knowledge for diagnostic companies it is important that research/academic laboratories developing Biosensing technologies, IVDs and POCs must be aware of these requirements early on to ensure translation of research into practice.

We Like it We Like it Not: Mass Spectroscopy for Biomarker Discovery

Mass spectroscopy (MS) in Biosensor Blog! Let me explain. Biosensors are routinely used in doctor’s clinic or as point-of-care devices to detect biomarkers for diagnosis and prognosis of diseases. There is however increasing demand for better clinical tests to detect diseases at early stage or to identify markers that may predict our predisposition to specific diseases. Hence, instead of testing for a single biomarker the trend is to identify panel of biomarkers that together may be a better predictor of a disease. That’s where MS is playing a very important role and have in fact become a standard tool for biomarker discovery. Advance MS techniques can detect upto 4000 proteins simultaneously in a single sample. In an ideal world we would compare samples from healthy individuals with samples from patients using MS and identify the up and down regulations of specific proteins that can then be used as biomarkers for diagnosis/prognosis of diseases. The now famous study by Petricoin and Liotta in 2002 did exactly that for ovarian cancer and claimed “—result yielded a sensitivity of 100% (95% CI 93–100), specificity of 95% (87–99), and positive predictive value of 94% (84–99)”. The techniques they used for their analysis was SELDI (surface-enhanced laser desorption and ionization) from Ciphergen. The claims from the study were disputed because of serious concerns about the reproducibility and reliability of the results and the Ciphergen has since then gone out of business and SELDI was bought by BioRad. The study however showed the power of MS for profiling the serum proteome and drove researchers to further exploit this powerful technology.

Fast forward 7 years and one would think that the problem of reproducibility and reliability would have been solved by now. But not so, going by couple of reports in past two months in high impact journals. I talked about the first report published in Nature Methods in April 2009 in my previous post. That study identified serious inter-laboratory reproducibility problems and attributed them mainly to environmental contaminations and deficiencies in databases used to identify proteins. Second paper that came last week in Nature Biotechnology had just the opposite conclusions! According to the report

“Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma.”

What gives? Turns out that the recent report differ from previous one in key aspects

  • Method used by the latest report is a quantitative method called multiple reaction monitoring (MRM) whereas previous one was qualitative method
  • Number of samples in recent study were 7 compared to 20 in previous one and
  • Number of laboratories in recent study were 8 compared to 27 in previous one
  • Protein concentrations in current study were moderately abundant >2ug/ml compared to 5pmol in previous study
  • There were Oother differences

But still completely different conclusions from two studies may be considered astounding for somebody looking at this technology to provide better and painless diagnosis during their next doctor’s visit. These studies show we still have some way to go before MS becomes an accepted tool for biomarker discovery. As I was reminded early in my research career that any new technology is 1% inspiration and 99% perspiration.

What this all has to do with the Biosensors? – Plenty! First, as a biomarker discovery tool MS is expected to provide a wealth of well characterized biomarkers that can then be validated and transferred to biosensor format for use in Doctor’s clinic. Second, biosensing is moving full ahead with complex technologies for multiplex sensing be it protein arrays, encoded bead based sensors or micro/nano-cantilever sensors. It is worth learning the lessons from MS that reproducibility/reliability across Laboratories/Operators/Instruments/Samples/Patients/etc. etc. should be front and center if any serious foray in diagnostics is intended.


Gold Nanoparticles for Cancer Cell Detection

When I posted my article on Gold Nanoparticles, my thoughts were that all the ‘major’ biosensing applications have been covered but it took less than 10 days for a cool new application to pop up. Prof Rotello at University of Massachusetts Amherst has developed a gold nanoparticle based chemical nose/sensor to “sniff out” not only cancerous cell from healthy cells but also differentiate between metastatic and non-metastatic cancer cells. At the core of this sensor are gold nanoparticle-fluorescent polymer dyads. Each of these dyads consists of gold nanoparticles that are designed to have a cationic surface charge conjugated with a fluorescent polymer. In these dyads, Gold nanoparticles quench the fluorescence of the polymers. In presence of cells, the cationic gold nanoparticles interact with cell surface (phospholipids, membrane proteins and carbohydrates) freeing up the polymer and dramatically increasing the fluorescence signal. After careful optimization, the researchers selected three gold nanoparticles with different surface groups that interact with polymer differently. When set of these three sensors are added to cells each gold nanoparticle interact with cells in different manner and there is net increase or decrease in fluorescence based on cell types. The array of three sensors was able to distinguish cancer cells from healthy normal cells and also the cancer cells that have metastasized or not.

The sensors obviously are working nicely in simulated laboratory condition but as many biosensor scientists already know the challenge is moving the system to real life situation. How the array sensor will work when injected into blood stream or a drop of blood is still need to be investigated. In complex biological systems cationic gold nanoparticles can potentially interact not only with any number of cells but also proteins hence contributing to large background signal. The quencher—polymer dyad idea has been around for at least a decade now. David Whitten in 1999 proposed a biosensor using similar approach. In addition, Alan J. Heeger (Winner of 2000 Nobel Prize in Chemistry) has developed biosensors using quenched fluorescent polymers. The lessons learned from previous work will hopefully make dealing with challenge little easier.

But idea of a simple method for detecting cancer cells in Doctor’s clinic is just too attractive that despite my serious reservations about the technology I will be rooting for it.