Test strip instead of enzyme immunoassay
MIPT nanobiotechnologists have placed a high-precision blood test in a conventional test strip
Researchers from IOF RAS and MIPT have developed a new biosensor test system based on the use of magnetic nanoparticles and designed to very accurately measure the concentration of protein molecules (for example, so-called "markers" that indicate the onset or development of diseases) in various samples, including opaque or strongly colored liquids. The study is published in the journal Biosensors and Bioelectronics.
The new development resembles a pregnancy test in its principle. The analysis is carried out using a small test strip made of porous material with two reaction lines. A drop of the test liquid is applied on one side; after a while, the result is manifested by the activation of either one or two lines. Such a test strip can be stored for a long time before use. The test takes little time, does not require specially trained personnel, can easily be carried out next to the patient and even in the field.
Magnetic nanoparticles are "crosslinked" with antibodies to the desired protein at the molecular level, and then they are applied to a porous plate near the intended place of contact with the studied solution. The liquid, spreading through the plate due to the capillary effect, captures magnetic particles. Then it meets two lines – test and control. The test contains antibodies that detain the protein of interest and at the same time those magnetic tags that are connected to the protein molecules due to the fact that the nanoparticles are also "crosslinked" with antibodies. And the control line only detains antibodies with magnetic tags, and it works in any case if the test strip is suitable for use. The role of the control line is an indicator that the test is usable, that the protein antibodies in its composition have not collapsed from improper storage and that the analyzed liquid has got to the right place.
After the sample has soaked the test strip and the antibodies have interacted with each other, the result can be read. This is where the similarity with the pregnancy test ends. In the case of a "classic" pregnancy test, the result can be either "yes" or "no". And the developed test allows not only to detect the presence of a protein with high sensitivity, but also to accurately determine its concentration. The accuracy of the determination will even exceed the accuracy of methods that are performed only in the laboratory and only by qualified personnel.
"Traditionally, tests that can be carried out not only in the laboratory, but even in the field, are based on the use of fluorescent or colored tags, and the results are determined visually, "by eye" or using a video camera," says the lead author of the study, researcher at IOF RAS Alexey Orlov (MIPT graduate 2010, graduated graduate school of MIPT in 2013). – We use magnetic particles, which have a significant advantage: with their help, you can perform an analysis, even by dipping a test strip into a completely opaque liquid, for example, to determine substances directly in whole blood. Precise numerical measurement is carried out strictly electronically using a portable device. Situations of "either yes or no" are absolutely excluded."
Scientists note that along with the high sensitivity of determining protein concentration, the new test system allows measurements to be carried out in a wide dynamic range: the upper threshold of the determined concentration exceeds the lower one by more than 4,000 times.
The term "dynamic range" is familiar to photographers: as applied to a camera, it denotes the ability of a matrix or film to distinguish brightness gradations without illumination and without turning the picture into a dark spot at all. For biochemical measurements, the dynamic range implies the ability to measure protein concentrations in both very dilute and very saturated solutions.
The operation of the new system was tested by measuring 0.025 nanograms per milliliter in the blood (at a rate of up to 4 nanograms) of prostate-specific antigen – one of the most massively controlled markers during the medical examination of men.
Prostate-specific antigen, PSA, is one of the possible markers of prostate cancer – and in criminology it is used to detect traces of seminal fluid. Both applications are associated with certain limitations and do not allow to unambiguously diagnose/prove the guilt of suspects, but the capabilities of the new biosensor platform are not limited to PSA analysis; this protein was chosen only for demonstration.
This sensitivity of the PSA determination is sufficient to understand whether a relapse has begun after the removal of the prostate gland, and the announced numbers give an idea of the development potential. It is able not only to show the output of any indicator beyond the norm, but also to easily track the dynamics of the concentration of protein markers of diseases.
Comparing the results obtained by the new method with the "gold standard" for determining PSA – enzyme immunoassay (ELISA), the scientists were convinced of the correctness of the new test system and its significant superiority over traditional technology.
In the new test system, the researchers applied their own patented MPQ (magnetic particle quantification) technique for highly sensitive counting of magnetic nanoparticles by nonlinear remagnetization, which allows registering from 60 zeptomoles (the prefix "zepto-" means 10 to minus twenty-first degree!) of nanoparticles in a linear range exceeding ten million times. The achieved parameters have no world analogues. The essence of the method consists in exposing nanoparticles to an alternating magnetic field at two frequencies and registering an induction response at combinatorial frequencies.
Many methods of substance analysis are based on the fact that the studied objects, whether particles or molecules, can be "rocked" by an electromagnetic field. At the right frequency, the sample begins to either actively absorb radiation or emit in response. In this case, the scientists used a combination of two magnetic field frequencies and monitored the response at a frequency that is their linear combination – this is called the "combinatorial" frequency.
"Previously, the high sensitivity of this method of detecting magnetic particles was demonstrated by us together with American researchers from the University of Chicago when our sensors registered magneto-radioactive nanoparticles based on the 59-Fe isotope in animals in vivo (see the work of M. Nikitin et al., J.Appl. Phys. 2008, 103, 07A304), – Maxim Nikitin, one of the co-authors of the study, head of the Laboratory of Nanobiotechnology at MIPT, comments on the creation of a new test system. – In particular, it was found that the detection threshold using the mentioned electronic method coincides with the registration threshold for concomitant gamma radiation, which makes it possible to replace radioactive tags with magnetic nanoparticles in a number of areas of biophysical research. In the current work, we use this methodology to achieve hypersensitivity already for immunoassay."
The phrase "the detection threshold using the mentioned electronic method coincides with the registration threshold for concomitant gamma radiation" means that magnetic tags and a special device for their detection can reveal all the same things that previously could be done with the help of radioactive preparations. From the point of view of physicians, magnetic preparations for diagnostic examination are obviously better, since they save patients from unnecessary radiation.
According to the head of research and head of the laboratory of IOF RAS Peter Nikitin (graduate of MIPT 1979), the developed magnetic methods and recorders for reading nanomarkers on test strips allow not only to ensure the mentioned limits and ranges of measurement of antigen concentrations, but also to effectively control all technological stages.
"In this way, we control the entire process: from the development and optimization of immunoassay protocols, to the implementation and interpretation of the results," says Peter Nikitin. – This, in particular, is achieved by quantitative monitoring of the redistribution of nanomarkers in the process of biochemical reactions along all components of 3D porous components of test strips, which has not been implemented so far by other methods. In addition, iron salts used for the synthesis of magnetic nanoparticles are incomparably more accessible and cheaper than reagents for the synthesis of gold nanoparticles, the most common in threshold tests such as pregnancy tests."
The combination of reliability, availability and high accuracy with high sensitivity allows us to count on the rapid transition of development from a laboratory prototype to mass production. The developers have not yet named specific dates, but emphasize that their test system can be used not only for the diagnosis of diseases, but also in a number of other tasks. The biosensor will allow for the analysis of food and medicines, with its help it will be possible to conduct environmental monitoring of the environment, and all this – right on the spot, without complicated and expensive devices.
A drop of the test liquid is applied to the strip (1). The liquid, spreading along the strip due to the capillary effect, captures magnetic particles that are "bonded" with antibodies to the analyzed protein (2). In the process of moving along the strip, the particles bind to the desired protein. Next, the fluid flow meets two lines – test (3) and control (4). The test line contains antibodies that delay the protein and at the same time those magnetic tags that are connected to it. The control line is triggered if the test strip is usable.
The analyzed protein (for example, PSA). MP is a magnetic nanoparticle hanging from below Y is an antibody to the analyzed protein. Test antibodies (blue Y) delay the analyzed protein, and control antibodies (yellow Y) – antibodies together with nanoparticles. Images provided by the MIPT press center.