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misconception of label-free detection

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. In 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 significant losses to GE Healthcare over the period of 10 years.  

By 2000-2002, Biacore approached very close to technical limits of SPR detection - created a benchtop machine with the 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.

For similar reason, label-free methods do not work in the range of concentrations that are clinically and diagnostically significant 10-15 -10-18 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. This is an emotionally attractive feature, which is behind the unrealistic dream of label-free. At first glance, there is nothing wrong in the idea to use intrinsic labels; they are already naturally contained in biological analytes. No need for labeling. The problem is that they are contained in myriad of irrelevant molecules of the matrix and similar biomolecules in huge amounts. Why these irrelevant intrinsic labels on other molecules create a problem? The problem is that these intrinsic labels do not allow to distinguish the desired target molecule of interest, protein, or nucleic acid, at the large background  of irrelevant homologs. Label-free advocates argue: “We will subtract the background.” The opponents respond: “You can subtract the background, but you cannot get rid of the background noise.” It appears that thermal noise of the background and its fluctuations caused by other molecular processes impose principle limitation to the limit of detection of label-free methods.

In the case of SPR, the intrinsic 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. It appears that the amplitude of  thermal fluctuations is always significantly larger than the anticipated signal from low concentration bioanalytes 10-15 -10-18 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 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 moves 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 failures 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), ~$20 billion on label-free. 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 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. 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.

ILabel-free methods appear to be not sensitive enough for molecular diagnostics. Sub-micromolar (10-7 10-8 mol/liter) level appears to be the lowest level of detection for 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 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. We believe that we need a “Journal of Failed Research” to prevent the waste of resources.

Misconception of Label-Free Biodetection

Synopsis. There is a die-hard misconception in the area of biodetection and molecular diagnostics, which erroneously assumes that “label-free” detection can be used for molecular diagnostic tasks that require a femto-molar (10^-15 M) and in certain cases atto-molar (10^-18 M) limits of detection. Indeed, several “label-free” methods have established themselves as convenient tools for detection of bioanalytes at micro-molar (10^-6 M), and in certain cases sub-micromolar levels of concentrations. It is erroneously assumed that the limit of detection of “label-free” methods can be improved down to femto-molar (10^-15 M) level. However, numerous attempts of multitude research groups have failed. Unfortunately, it is not a common practice to publish the results of failed attempts. Our own data on “label-free” detection, analysis of small portion of data obtained by other groups, and premises based on principles of statistical physics show that “label-free” methods, unfortunately, cannot be used for detection of low abundance molecular marker, which is necessary for diagnostics.

Introduction. 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. However, experimental data and theoretical premises assume that there are insurmountable limitations imposed by the principles of statistical physics for pushing the limit of detection beyond the micro-molar (10^-6 mol/liter) level.

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 in conflict with basic law of physics. Nevertheless, each year the U.S. Patent and Trademark Office 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 to detect biomolecules with concentrations down to femptomolar, and in certain cases - attomolar levels (10-15 10-18 mol/liter). Many of established and emerging molecular markers operate at the range of very low concentrations 10-15 -10-18 mol/liter. The purpose of this web page is to draw your attention to the fact that all known “label-free” detection systems are not capable of attaining the level of detection 10-15 -10-18 mol/liter. Numerous attempts to attain the necessary sensitivity and limit of detection have failed. It appears that there are limitations imposed by basic principles of physics that do not allow to improve the limit of detection of label-free methods to the necessary level.  

Who should read this web page? We believe that the entire biodetection community should be aware of the facts that the idea of label-free diagnostics has been tried by numerous groups; all attempts failed. U.S. Government officials, who solicit applications for respective grants should read this page. It appears that there are obstacles imposed by basic principles of physics. Before you start your own attempt, be aware that systematic attempts of numerous research groups have failed. Unfortunately, most of negative results remain unpublished.  

We estimate that the U.S. Federal Government has wasted estimated $20 Billion to entertain the idea of label-free detection. The U.S. Government lacks a mechanism, which would require to publish the results of failed attempts in public domain. As a 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 that label-free diagnostics is possible. Such erroneous message issued by reputable NIH and NSF not only distracts funds to a dead-end efforts, but also damages the entire landscape and 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 one more erroneous idea of label-free diagnostics. Dr. Andrew Conrad, from Google, tried to sell to mass media a Star-Track 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 premises 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 all label-free methods.

Not surprisingly, search of the literature using NIH’s PubMed and other databases gives no results for label-free molecular diagnostics. The multitude attempts and wasted ~$20 billion did not result in a label-free technology, suitable for molecular diagnostics.

On the other hand, label-based methods have established themselves as indispensable tools for molecular diagnostics. 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.