CC BY
14B-I-16
Highly sensitive optical methods for differential diagnosis of autoimmune diseases and identification of DNA molecules
P.I. Nikitin1, A.V. Orlov1, V.A. Bragina1, A.V. Pushkarev12, E.N. Mochalova12,
M.P. Nikitin2, B.G. Gorshkov1
1- Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilov St., Moscow 119991,
Russia
2- Moscow Institute of Physics and Technology, 9 Institutskii per., Dolgoprudny, 141700, Russia
Email: nikitin@kapella.gpi.ru
Highly sensitive optical methods are developed for differential diagnosis of autoimmune diseases based on the spectral-correlation interferometry [1] and image processing of a microarray glass sensoi chip. The methods are based on real -time label-free measurements of changes in the thickn ess of biolayers formed by target and recognition molecules on the surface of a sensor chip.
The related biosensing system has been tested by measuring in human serum both critical characteristics of autoantibodies: concentration and native kinetic parameters that reflect autoantibody aggressiveness to the organism's tissues. Rapid (<25 min) and simultaneous characterization of several autoantibodies in the same serum sample has been demonstrated for anti-thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti -TPO). The biosensor offers extremely high sensitivity: the limits of detection in serum are 1.7 and 6 IU/ml for anti -TPO and anti -TG, respectively. The dynamic range covers the whole range of clinically relevant concentrations.
New criteria in comprehensive diagnostics of autoimmune diseases has been proposed for the first time based not only on traditional measurements of concentration of autoantibodies, but also on quantitative evaluation of autoantibody aggressiveness.
The developed interferometric instruments have been also successfully used for biosensing based on glass sensor chips coated with a graphene layer functionalized with aptamers, which specifically bind low molecular weight compounds, e.g., Ochratoxin A [2].
Besides, the biosensor systems have been tested for counting magnetic nanobioconjugates, which were used simultaneously as carriers for magnetic separation of target molecules and as biospecific nanolabels that amplify the recorded optical signal.
A novel rapid method has been developed for accurate characterization of bioconjugates based on nanoparticles and determination of kinetic properties of their binding with target molecules . A mathematical model has been elaborated to describe analytically this binding process [3]. The method performance has been demonstrated by detection of protein enterotoxins (staphylococcal enterotoxin B) and biomarkers of cardiac diseases (cardiac troponin I).
Synthesized nanoparticles conjugated with biorecogni tion molecules have been also used for development of rapid formats of in vitro immuno- and DNA assays. In particular, a new ultrasensitive method is developed for direct (without amplification of reaction) mea suring the concentration and identification of RNA / DNA molecules based on a combination of single-stranded RNA fragments anc gold nanoparticles . The record sensitivity up to 30 fM is achieved for DNA concentration in an extremely small sample volume of 2 0 ^l with a rapid (15 min) and easy -to-perform
immunochromatographic assay [4]. The achieved detection limit at such sample volume is on the level of a typical yield of target DNA from a biopsy sample (0.5 amol o 3 * 105 molecules). That is promising for development of new generation tools for diagnosing diseases.
[1] A.V. Orlov, A.V. Pushkarev, S.L. Znoyko, D.O. Novichikhin, V.A. BraginaB.G. Gorshkov, P.I. Nikitin, Multiplex labe-free biosensor for
detection of autoantibodies in human serum: tool fornew kinetics-based diagnostics of autoimmunediseases, Biosensors & Bioelectronics vol. 159, p. 112187 (2020).
[2] N. Nekrasov, N. Yakunina, A. V. Pushkarev, A. V. Orlov, I. Gadjanski, A. Pesquera, A. Centeno, A. Zurutuza, P.I. Nikitin, Bobrinetskiy Spectral-Phase Interferometry Detection of Ochratoxin A via Aptamer-Functionalized Graphene Coated Glass, Nanomaterials, vol. 11, p 226 (2021).
[3] A.V. Pushkarev, A.V. Orlov S.L. Znoyko, V.A. Bragina, P.I. Nikitin. Rapid and easy-to-use method for accurate characterization of targel
binding and kinetics of magnetic particle bioconjugates for biosensing Sensors, vol. 21, p. 2802 (2021).
[4] V.R. Cherkasov, E.N. Mochalova, A.V. Babenyshev, A.V. Vasilyeva, P.I. Nikit, M.P. Nikitin, Nanoparticle beacons: supersensitive smar
materials with on/off switchable affinity to biomedical targets, ACS Nanq vol. 14, pp. 1792-1803 (2020).
