1 Institute of Biomedical Chemistry, Moscow, Russia
2 Joint Institute for High Temperatures of Russian Academy of Sciences, Moscow, Russia
3 Rzhanov Institute of Semiconductor Physics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia
4 N.N. Blokhin National Medical Research Center of Oncology, Moscow, Russia
5 Foundation of Perspective Technologies and Novations, Moscow, Russia
6 JSC Novosibirsk Plant of Semiconductor Devices with OKB, Novosibirsk, Russia
The detection of CA 125 protein in buffer solution with a silicon-on-insulator (SOI)-based nanoribbon (NR) biosensor was experimentally demonstrated. In the biosensor, sensor chips, bearing an array of 12 nanoribbons (NRs) with n-type conductance, were employed. In the course of the analysis with the NR biosensor, the target protein was biospecifically captured onto the surface of the NRs, which was sensitized with covalently immobilized aptamers against CA 125. Atomic force microscopy (AFM) and mass spectrometry (MS) were employed in order to confirm the formation of the probe–target complexes on the NR surface. Via AFM and MS, the formation of aptamer–antigen complexes on the surface of SOI substrates with covalently immobilized aptamers against CA 125 was revealed, thus confirming the efficient immobilization of the aptamers onto the SOI surface. The biosensor signal, resulting from the biospecific interaction between CA 125 and the NR-immobilized aptamer probes, was shown to increase with an increase in the target protein concentration. The minimum detectable CA 125 concentration was as low as 1.5 x 10−17 M. Moreover, with the biosensor proposed herein, the detection of CA 125 in the plasma of ovarian cancer patients was demonstrated.
Keywords: ovarian cancer; nanoribbon biosensor; silicon-on-insulator; CA 125; aptamers; surface functionalization
The effectiveness of cancer therapy depends on the stage at which the disease is revealed; timely revelation of cancer facilitates the treatment and improves its effectiveness [1,2]. This is why early revelation of target cancer-associated biomarkers is required. The blood concentration of the majority of proteins, including the known disease markers, is low (<10−13 M) . Particularly, Rissin et al. emphasized that at an early stage of cancer in humans, the blood concentration of cancer-associated marker molecules is at femtomolar (10−15 M) levels . At the same time, the sensitivity of methods commonly employed in modern clinical practice — such as enzyme-linked immunosorbent assay (ELISA)-based approaches — is much lower, only allowing the achievement of 10−12 M to 10−7 M detection limits . Another disadvantage of these methods consists of the use of additional labels (such as enzymatic or fluorescent ones).
Biosensor-based detection represents an approach commonly used for the characterization of intermolecular interactions , which is applied for biomarker detection . The use of biosensors with sensor elements of nanometer size — such as nanowires and nanoribbons — opens new horizons in biomedical research. These biosensors allow one to perform direct label-free detection of various targets — such as viral particles  and biological macromolecules (proteins [7,8,9,10,11,12,13] and nucleic acids [14,15,16,17,18,19])—in real time, attaining low (<1 fM, i.e., <10−15 M) concentration detection limits . The principle of operation of these biosensors consists of recording a modulation of an electric current, flowing through the sensor elements, in the course of binding of target particles (i.e., macromolecules or viral particles of interest) to their surface. The surface-bound molecules play the role of a virtual gate, while the sensor structure itself represents a field-effect transistor (FET) of nanometer size . Since the sensor elements are characterized by a high surface-to-volume ratio , a very high sensitivity of the nanoribbon (NR)-based devices towards the target particles is achieved. In theory, the detection limit of a single target particle per individual sensor element is attainable . For viral particles, the single-particle sensitivity of a nanowire biosensor was experimentally demonstrated by Patolsky et al. . For biological macromolecules, femtomolar and even sub-femtomolar detection limits were attained for nucleic acids [15,23] and for proteins [11,20], respectively. In this way, the data reported in the literature clearly demonstrate that nanoribbon biosensors represent very attractive tools for highly sensitive biomarker detection in both research and clinical practice.
Biological molecules are known to express their function through interactions with other molecules . Hence, in a biosensor device intended for clinical applications, biospecific detection of target molecules should be provided. For this purpose, the surface of the sensor elements is functionalized with molecular probes, which are able to selectively bind the target molecules . Sometimes, the sensor chip represents a surface bearing molecular probes against only one target molecule . For clinical applications, however, multiplexed detection is required in order to provide simultaneous detection of several different biomarkers in one sample [7,26]. In the latter case, the single sensor chip contains an array of multiple sensor elements, and each sensor element is sensitized with molecular probes against one distinct target; this is another advantage of nanowire and nanoribbon biosensors [7,8,26].
For the sensitization of the sensor elements, aptamers [11,13] and antibodies [5,8,12] are commonly employed. The time stability and chemical resistance of antibodies are poor, while their production cost is high. In contrast, aptamers, which represent single-stranded DNA or RNA oligonucleotides capable of biospecific binding with target molecules, are characterized by higher chemical and time stability [27,28] and relatively low production costs, thus being devoid of the disadvantages typical of antibodies.
Ovarian cancer ranks fifth in cancer deaths among women, being the cause of more deaths than any other cancer of the female reproductive system. A woman’s risk of getting ovarian cancer during her lifetime is about 1 in 78 . In terms of one-year survival rate, 98% of patients diagnosed at stage I survive their disease—compared with 53.8% of patients diagnosed at stage IV . This clearly indicates why the early diagnosis of ovarian cancer represents an urgent problem in medicine. The discovery of carbohydrate antigen 125 (CA 125) has become a key step on the path towards the non-invasive diagnosis and monitoring of ovarian cancer. CA 125 represents a glycoprotein epitope of a high-molecular-weight mucin . Since CA 125 was suggested as a marker of ovarian cancer in 1983, this protein represents a benchmark for monitoring ovarian cancer patients .
Biosensor-based detection of CA 125 has recently been reported in a number of papers. Hence, Szymanska et al.  developed a surface plasmon resonance imaging (SPRI) biosensor for the label-free detection of CA 125 in human serum; in this biosensor, polyclonal antibodies against CA 125 were employed as molecular probes; however, no real-time measurements were reported in this paper . Mandal et al.  reported the fabrication of an electrochemical (carbon nanotubes)-based biosensor, in which monoclonal antibodies against CA 125 were utilized as molecular probes; the authors, however, performed their experiments at a rather high (560 μg/mL) concentration of CA 125. Attia et al.  developed a luminescence-based assay for the detection of CA 125 in serum; in this assay, acridinium-ester-labeled monoclonal antibodies were employed as molecular probes, and fluorescein-labeled monoclonal antibodies were used as secondary antibodies—that is, additional labels were employed. Petrova et al.  developed an interesting photonic-crystal-based biosensor for the multiplexed, label-free, real-time detection of CA 125 and two breast cancer markers (human epidermal growth factor receptor 2 and cancer antigen 15-3). Monoclonal antibodies against the target biomarkers were used as probe molecules . It should be emphasized that, in contrast to aptamers, antibodies are quite expensive, while exhibiting poorer chemical and time stability, thus leading to the high cost of the assay.
In the present study, a nanoribbon biosensor (NR biosensor) was employed for the detection of CA 125 in buffer solution. The sensor chip, bearing an array of 12 separate nanoribbons, was fabricated on the basis of “silicon-on-insulator” (SOI) structures of n-type conductance. SOI structures represent precisely engineered multilayer semiconductor/dielectric structures, and the technology of fabrication of nanoribbon sensors, based on these structures, was reported by Naumova et al. ; the use of this type of structure in advanced silicon devices is promising. SOI structures are fabricated using a complementary metal–oxide–semiconductor (CMOS)-compatible technology . The latter makes our technology of sensor chip fabrication very convenient, since it allows for production scaling and for integration of the NRs with additional electronic circuitry intended for signal processing . The surface of individual NRs was sensitized with aptamers against CA 125, which were used as molecular probes. Atomic force microscopy (AFM) and mass spectrometry (MS) were employed in order to confirm the efficiency of the technique used for the sensitization of the NRs. Such an approach was shown to be an efficient tool in studying antibody–antigen  and aptamer–antigen [39,40] complexes on the surface of solid substrates sensitized with molecular probes. Since immobilized aptamers are barely distinguishable on the AFM substrate surface, the efficiency of the sensitization of the substrate surface with aptamers is estimated after the formation of aptamer–antigen complexes on it . Via AFM and MS, the successful formation of aptamer–antigen complexes on the surface of SOI substrates with covalently immobilized aptamers against CA 125 was revealed. This fact confirms the sufficient efficiency of the NR sensitization technique employed. By using the NR biosensor with aptamer-sensitized sensor elements, successful detection of CA 125 at ultralow concentrations was demonstrated, and the CA 125 concentration detection limit was as low as 20 aM (2 x 10−17 M). This value is an order of magnitude lower than that obtained with the use of antibodies against CA 125 as molecular probes . Successful use of the SOI-based NR sensor chips for the revelation of CA 125 in plasma of ovarian cancer patients was been demonstrated. This indicates that the NR biosensor represents a promising tool for clinical applications.
Ivanov, Y.D.; Malsagova, K.A.; Pleshakova, T.O.; Galiullin, R.A.; Kozlov, A.F.; Shumov, I.D.; Popov, V.P.; Kapustina, S.I.; Ivanova, I.A.; Isaeva, A.I.; Tikhonenko, F.V.; Kushlinskii, N.E.; Alferov, A.A.; Tatur, V.Y.; Ziborov, V.S.; Petrov, O.F.; Glukhov, A.V.; Archakov, A.I. Aptamer-Sensitized Nanoribbon Biosensor for Ovarian Cancer Marker Detection in Plasma. Chemosensors 2021, 9, 222. https://doi.org/10.3390/chemosensors9080222