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Misconception of Label-Free Biodetection
Synopsis. There is a die-hard misconception of “label-free” detection in the area of biodetection and molecular diagnostics. It appears that label-free molecular diagnostics is an unrealistic dream. Quartz Crystal Microbalance (QCM), Surface Plasmon Resonance (SPR), field effect transistors (FET), microcavity laser resonators, nanowires, and other methods that use intrinsic labels (mass of molecules - QCM, refractive index - SPR, electrical charge -FET) are capable of detecting micro-molar (10^-6 M), and in certain cases sub-micromolar concentrations of bioanalytes. However, for biodetection and molecular diagnostics tasks a femto-molar (10^-15 M) and in certain cases atto-molar (10^-18 M) limits of detection are necessary. Numerous failed attempt and preliminary estimates based on laws of statistical physics show that label-free methods, unfortunately, cannot be used for detection of low abundance molecular marker, which is necessary for diagnostics.
Science is a human endeavor and, therefore, is a subject to emotions, dreams, and sometimes die-hard sincere errors - misconceptions. The dream about a perpetual motion machine is an example of such die-hard misconceptions. Back in 1494, Leonardo da Vinci wrote: “Oh ye seekers after perpetual motion, how many vain chimeras have you pursued? Go and take your place with the alchemists.” Since then, fundamental laws of physics have confirmed that the dream about perpetual motion machine is nothing, but a dream. Nevertheless, each year the U.S. Patent and Trademark Office still receives dozens of applications for invention of perpetual motion machines.
There is a similar die-hard misconception in the area of biodetection - the dream about label-free detection. Unlike the perpetual motion, the idea of label-free detection is not prohibited by hard science; it does not contradict to fundamental laws of physics. Moreover, label-free methods have been successfully applied for the detection of biomolecules with concentrations down to micro-molar level (10-6 mol/liter). For molecular diagnostics, however, one needs a biodetection methods that are capable of measuring concentration of biomolecules down to femptomolar - tens of attomolar levels (10-15 10-17mol/liter). Many of established and emerging molecular markers operate at the range of very low concentrations 10-15 -10-17 mol/liter. The purpose of this web page is to draw your attention to the fact that label-free detection, in principle, is not capable of attaining the level of detection 10-15 -10-17 mol/liter, and therefore is not suited for accurate molecular diagnostics. Below, we will provide preliminary data and arguments that the idea of label-free molecular diagnostics is an unrealistic dream.
Who should read this web page? We believe that the entire biodetection community should be aware that the idea of label-free diagnostics is not realistic and based on misconception. We estimate that the U.S. Federal government has wasted estimated $20 Billion to entertain the idea of label-free detection. Unfortunately, the U.S. Federal government lacks a mechanism, which would require to document failed attempts of label-free detection. As an unfortunate result of this deficiency, National Institutes of Health (NIH) and National Science Foundation (NSF) continue to solicit grant applications for label-free detection, sending to research community the erroneous message of allegedly possible label-free diagnostics. Such erroneous message issued by reputable NIH and NSF is damaging for the future of molecular diagnostics. This error propagates to other agencies and private investors and changes the landscape for decades. Privately funded labs, such as Google’s labs, Gates’ Foundation, and other private and public firms typically are stuffed with scientists and administrators, who depended in the past, or will depend in the future on NIH and NSF. Not surprisingly, Google has recently came up with an anti-scientific erroneous idea of nanoparticles for diagnostics. Dr. Andrew Conrad, from Google, tried to sell to mass media his erroneous idea of label-free detection using nanoparticles circulating in blood. His attempt caused rational criticism expressed by biodetection experts. The damage, however, has been done. Even in our educated age, the error will propagate in time and space. We believe that it is important to present correct scientific information about the erroneous misconception of label-free detection. In TIRF Labs we have accumulated significant body of experimental evidence against the idea of label-free diagnostics. We also assembled a body of qualitative data and ideas that suggest that the thermal noise of large background signal, which is inherent to all label-free methods, impose serious limitations on the limit of detection of label-free methods. Not surprisingly, search of the literature using NIH’s PubMed and other databases gives no results for label-free molecular diagnostics. On the other hand, there are thousands of published scientific papers that have documented the ultimate limit of detection - single molecules - for the case of label-based methods. We invite theoreticians proficient in the area of statistical physics to support theoretically the fact of insufficient sensitivity of label-free methods. It appears that a theoretical approach similar to the Smoluchowski equation, Fokker-Plank theory, and Kolmogorov forward and backward equations can be employed to theoretically predict the best limit of detection for label-free methods. Before this has been done, we offer to your attention our qualitative consideration of the label-free methods.
Consider the principles of label-free biodetection. Surface Plasmon Resonance (SPR) provides an example of tremendous efforts and dramatic failures in the area of label-free. Let us take a closer look at SPR biodetection. One of the leaders in SPR instrumentation is Biacore, a company founded in 1984 under the name of Pharmacia Biosensor AB, by Pharmacia and the Swedish Defence Research Agency. In 1996 the company changed its name to Biacore AB Corporation. The first Biacore SPR instrument was issued in 1990. n June 2006, GE Healthcare acquired Biacore for $390 million. By 2006, Biacore has saturated the market with expensive SPR instruments. It appears that the acquisition resulted in losses to GE Healthcare over the period of 10 years.
By 2000-2002, Biacore approached very close to theoretical limit of SPR detection - created a benchtop machine with price tag ~$500,000 (approximately half of million US dollars). The instrument possessed analytical limit of detection at submicromolar (~10-7 mol/liter) level of concentrations. This limit appears to be close to that of theoretically possible. Typically, SPR instruments are capable of detecting down to micro-molar concentrations (10-6 mol/liter). In rare cases, SPR can detect down to sub-micromolar levels (~10-7 mol/liter). However, the diagnosing of pathogens and diseases requires, as mentioned above, more sensitive tests - capable of detecting femtomolar (10-15 mol/liter) and attomolar (10-18 mol/liter) concentrations. The concentrations of protein, nucleic acid, and metabolite molecular markers should be measured in complex matrix of biological fluids, such as blood, full of other proteins and nucleic acids. The task of biodetection for diagnostic purposes is frequently compared to the challenge of finding a needle in a haystack. In fact, the task of biodetection is more challenging - it is like finding a unique straw with a specific shape among myriad of similar straws in the haystack. If the straw of interest is not labeled (or its counterpart - specific affinity reagent - detection antibody or probe DNA are not labeled), your chance to find the target among myriad of similar molecules is close to zero.
Unfortunately, label-free methods do not work in the range of concentrations that are clinically and diagnostically significant 10-15 -10-17 mol/liter. Why? Let us take a look into the principles of label-free. In fact, Surface Plasmon Resonance (SPR), Quartz Crystal Microbalance (QCM), field effect transistors (FET), microcavity laser resonators, nanowires, and all other allegedly “label-free” methods do use certain labels. The problem is that these labels are intrinsic. They are naturally contained in biological analytes. In huge amounts. The problem is that they are contained in myriad of irrelevant molecules of the matrix and similar biomolecules. Why this is a problem? These intrinsic labels are not specific - they do not allow to distinguish the desired target molecule of interest, protein, or nucleic acid, at the large background of irrelevant homologs. In the case of SPR, the label is refractive index, in the case of QCM - mass, FET - charge of molecules. The problem is that, similar to straws in the haystack, similar labels are present everywhere around the bioanalyte molecule of interest. Irrelevant labels create overwhelmingly large background. Advocates of label-free argue: “we will subtract the background”. Indeed, in certain cases you can subtract the background. But the worst problem is that the background fluctuates unpredictably. The “killer” news is that the amplitude of thermal fluctuations is larger than the anticipated signal from low concentration bioanalytes 10-15 -10-17 mol/liter. Our preliminary data suggest that label-free methods, in principle, are not capable of detecting femtomolar (10-15 mol/liter) and attomolar (10-18 mol/liter) concentrations of biomarkers.
Preliminary estimate shows that in the case of SPR only thermal fluctuations of the SPR background signal correspond to the limit of detection at- nanomolar (10-9 mol/liter) level. This estimate is supported by our own experimental data and by analysis of the vast literature on label-free detection. Similar estimates are valid for QCM, field-effect transistors, resonance laser cavities, carbon nanotubes, and similar methods that do not require specific extrinsic labels neither at the target molecule, nor at the affinity reagent molecule, e.g. antibody.
In practical systems, in addition to thermal fluctuations there are many other processes that contribute to fluctuations of the background signal. The cumulative effect of these undesirable fluctuation pulls the limit of detection to worse. For example, in the case of SPR, large changes in the background are caused by variations of the ionic strength and other parameters of the solvent that are related to incremental change of the refractive index. Without specific, distinguishable labels, there is no way to differentiate between the SPR signal caused by binding of the target molecule and that caused by thermal fluctuations and other irrelevant processes. Our preliminary analysis shows that similar estimates are valid to all other label-free methods. Thus, a nanomolar (10-9 mol/liter) level appears to be the lowest theoretical limit of detection for all label-free methods. In practice, many additional processes, such as mentioned above changes of matrix composition, non-specific interactions with the affinity reagents, temperature, ionic strength, natural and method-specific fluctuations in solvent and solute, cause additional unpredictable fluctuations of the signal. In practical instrument, label-free methods are capable of providing the limit of detection at micromolar or sub-micromolar levels 10-6 10-7 mol/liter.
There have been numerous failed attempt of using label-free for molecular diagnostics. The failure of these attempts indirectly support our assumption that label-free detection is not suited for molecular diagnostics. After 9-11, followed by the anthrax letter attacks, the U.S. government spent over $70 billion on biodefense, including estimated $40 billion on molecular diagnostics (MDx). Naturally occurring diseases, such as avian flu, SARS, Ebola, can be even more damaging than a man-made bioweapon. Damages caused by pathogens and toxins can be enormous. 9-11 anthrax letters caused economical and societal damages comparable with the effect of a nuke explosion in the Washington D.C. area. Countermeasures, prevention, decontamination, and retaliation require rapid and accurate MDx. Inaccurate or slow results of analyses are incredibly damaging and expensive. In 2002, false positive and false negative results of traditional MDx methods almost shut down the Winter Olympics in Utah. In the period 2001-2016 false positive and false negative results of tests seriously threatened many high-profile political events and caused damages measured by billions of dollars.
After 2001, billions of dollars were invested in the development of advanced MDx. Not surprisingly, there is no analytical technique, which has not been tried for MDx. In 2003-2004, the U.S. Department of Defense carefully analyzed all existing and emerging biodetection techniques. Among these techniques were so-called label-free methods, such as SPR, QCM, field-effect transistors, resonance laser cavities, carbon nanotubes, and similar methods. All these methods that do not require extrinsic labels neither at the target molecule, nor at the affinity reagent, failed. As mentioned above, the intention of “label-free” detection stems from our natural desire to have the results rapidly, inexpensively, and with minimal labor. However, in certain cases, as a proverb says: “Nice intentions pave the road to hell.” Nice intentions of humans sometimes disagree with laws of the nature. In particular, the intention to employ so-called label-free methods have failed. Numerous times. Nevertheless, new attempt are being made. We are stepping on the same rake, again and again. Billions of dollars have been wasted. This waste of resources and propagation of error of label-free detection should be brought to the end. We hope that posting of this web page will help to rectify the situation.
In principle, label-free methods can NOT be sensitive enough for molecular diagnostics. Sub-micromolar (10-7 10-8 mol/liter) level appears to be the lowest level of detection for all label-free methods, which is not sufficient for molecular diagnostics. We believe that the misconception of label-free should be brought to the daylight and actively discussed by the entire biodetection community. We believe that such discussion will facilitate the progression of highly efficient biodetection technologies, such as iDiagnostics.
If you have thoughts and opinions about the label-free detection and the situation in MDx area in general, please share them with us. Together, we can improve the situation and accelerate the progress in MDx. Advanced MDx technologies have been developed; they do use labels. However, some of the technologies detect unlabeled analytes.
15 years and $70 billion after 9-11, accurate and rapid MDx products are not at the market yet. Accurate and inexpensive molecular diagnostics devices such as iDiagnostics, might be in medical cabinets by now, if the label-free misconception and other misfortunate aspects would not hamper the progress. Precision diagnostic devices and methods have been developed. However, they are still in laboratories, including TIRF-EC Microarray and iDiagnostics in TIRF Labs. Meantime, their way to the general public is blocked by the label-free misconception. Billions of dollars of the U.S. federal funds continue to be wasted for useless attempts. Why the results of failed attempts have not been published? Why the U.S. government agencies continue to solicit label-free MDx? We believe that we need a “Journal of Failed Research” to prevent stepping again on the same rake.