RU2546518C2 - Method for analysing membrane-bound red cell haemoglobin by giant combined diffusion spectroscopy on nanostructured coatings - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: membrane-bound red cell haemoglobin by giant combined diffusion (GCD) spectroscopy is analysed by the use of nanostructured coatings in the form of ring silver nanostructures having the hierarchical structure. Silver ring rims consists of connected micron-scale porous silver aggregates, the surface of which comprises rounded silver nanoparticles 2-100nm embedded into a matrix. The red cell immobilisation time on the nanostructured coatings makes 5-40 minutes, and GCD spectra are generated by green laser at wave length 514 or 532 nm.

EFFECT: invention enables diagnosing membrane-bound haemoglobin in undamaged red cells by the giant combined diffusion.

4 cl, 4 ex, 1 dwg

Description

Technical field

The invention relates to the field of medical diagnostics and bioanalytical studies, can be used to detect biological molecules and markers in biological fluids (blood, saliva, etc.), study the structure of biomolecules, for forensic purposes.

State of the art

(Luneva, O.G., et al., Ion transport, membrane fluidity and haemoglobin conformation in erythrocytes from patients with cardiovascular diseases: Role of augmented cholesterol. Pathophysiology, 2007. 14(1): p. 41-46). Перечисленные факты дают возможность предположить, что уже на ранних этапах болезни существуют изменения в ионном составе цитоплазмы в примембранном пространстве эритроцитов, а также увеличивается число мест связывания Гб на плазматической мембране. Поскольку конформация Гб и его O₂-связывающие свойства напрямую зависят от концентрации ионов и связывания на мембране, то можно предположить, что по изменениям конформации Гб_мс можно судить о ранней стадии возникновения ССЗ. Таким образом, исследование функционального состояния и конформации Гб_мс имеет большое значение как для понимания процессов, протекающих в эритроцитах, так и может служить основой для проведения медицинской диагностики ССЗ.Giant Raman spectroscopy (GCR spectroscopy) using silver-based nanostructured materials is used in a wide variety of fields related to the detection of single cells and biological molecules at nanomolar concentrations. An important area of biomedical diagnostics using SERS spectroscopy is the study of blood, namely changes in the structure, conformation and, as a consequence, the oxygen-binding properties of hemoglobin (GB) in living red blood cells. The conformation and properties of GB can be mediated by pathologies of the cardiovascular system or a number of hereditary diseases. Erythrocytes are known to contain two types of hemoglobin: cytoplasmic (GB _cyt ) and membrane-bound GB (GB _ms ). Conventional Raman spectroscopy in blood tests allows recording Raman spectra only from GB _cyt in red blood cells, because the contribution of GB _{ms is} insignificant due to the fact that its concentration is very small - it is less than 0.5% of the concentration of GB _cyt . In the case of SERS spectroscopy, the amplification of the Raman signal comes from GB _ms located in close proximity to silver nanostructures, and thus, changes in the conformation and O ₂ -binding properties of GB _ms can be estimated from the Raman spectra. With many cardiovascular diseases (CVD) and diabetes mellitus, the protein composition and the submembrane cytoskeleton of erythrocytes are known to change and the amount of protein of band 3 (anion exchanger AE1) increases, to which a membrane-bound GB _ms is attached (Santos-Silva, A., et al ., Erythrocyte damage and leukocyte activation in ischemic stroke. Clin. Chim. Acta 2002. 320 (1-2), p.29-35 and Petropoulos, IK, et al., Structural alterations of the erythrocyte membrane proteins in diabetic retinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 2007,245 (8), p. 1179-1188); in CVD and diabetes, the activity of ion transport through the plasma membrane of erythrocytes changes, and therefore, in the submembrane space of red blood cells, the local concentrations of ions, primarily H ⁺ , Ca ²⁺ ions, change most significantly,

(Luneva, OG, et al., Ion transport, membrane fluidity and haemoglobin conformation in erythrocytes from patients with cardiovascular diseases: Role of augmented cholesterol. Pathophysiology, 2007.14 (1): p. 41-46). These facts make it possible to assume that already in the early stages of the disease there are changes in the ionic composition of the cytoplasm in the near-membrane space of red blood cells, and the number of GB binding sites on the plasma membrane also increases. Since the conformation of GB and O ₂ -binding properties directly depend on the concentration of ions and binding to the membrane, it can be assumed that changes conformation GB _ms can judge the early stages of CVD. Thus, the study of the functional state and conformation of GB _ms is of great importance both for understanding the processes occurring in red blood cells and can serve as the basis for medical diagnosis of CVD.

A known method of studying GB _ms in intact erythrocytes by Raman spectroscopy (Brazhe, NA, et al., New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy. Biophysical Journal, 2009. 97 (12): p. 3206-3214) . In this work, silver nanoparticles (NPs) obtained by the Leopold and Lendl method were mixed with red blood cells in a 1: 1 ratio to a homogeneous mixture. The SERS spectra were recorded using a green laser with a wavelength of 532 nm. The estimated gain for different absorption bands of the hemoglobin porphyrin ring are: in the region of 1172 cm ^-1 -1.3 · 10 ⁵ , 1375 cm ^-1 -1.5 · 10 ⁴ , 1580 cm ^-1 -2.9 · 10 ⁴ , 1640 cm ^-1 -1.4 · 10 ⁴ .

An analogue of the present invention is a method for analyzing β-carotene and hemoglobin in human blood plasma using giant Raman spectroscopy (Casella, M., et al., Raman and SERS recognition of β-carotene and haemoglobin fingerprints in human whole blood. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2011. 79 (5): p. 915-919).

A significant drawback of these methods for studying blood plasma and GB _ms in erythrocytes is the use of a colloidal solution of nanoparticles (NP), which can have toxic and osmotic effects on cells, mask the HRS signal with organic molecules or by-products that are part of NP sols. In addition, the absence of effective direct contacts of NPs with blood plasma cells or biomolecules is possible. These problems are successfully solved in the claimed invention using nanostructured coatings in the form of ring silver nanostructures having a hierarchical structure.

A method for studying blood plasma and red blood cells using SEC spectroscopy (Premasiri, WR, J.C. Lee, and LD Ziegler, Surface-Enhanced Raman Scattering of Whole Human Blood, Blood Plasma, and Red Blood Cells: Cellular Processes and Bioanalytical Sensing The Journal of Physical Chemistry B, 2012. 116 (31): p. 9376-9386). In this work, a drop of blood preliminarily mixed with gold nanoparticles was deposited on a SiO ₂ substrate and allowed to dry for 1 h. The SERS spectra were recorded using a 785 nm laser. A distinctive feature of the above method from the claimed invention is that the SQR spectra are not taken from living red blood cells, but from a dried drop of blood. In this regard, this method cannot be used for medical diagnostics of living bioobjects.

The only work in which nanostructured substrates were used for SERS analysis of living cells was the work on detecting bacteria in the blood (Liu, TY, et al., Functionilized arrays of Raman-enhancing nanoparticles for capture and culture-free analysis of bacteria in human blood. Nature Communications. 2011.2: 538. DOI: 10.1038 / ncomms1546). In this work, it was proposed to use substrates based on anodic alumina, decorated with an array of silver nanoparticles and coated with a layer of vancomycin. The lack of such substrates for the multifunctional use of biochips is determined by the need to replace the vancomycin layer with other glycopeptides in order to detect other microorganisms. In addition, such substrates cannot be used to study non-bacterial cells.

Thus, analogues of the present invention known from the prior art do not allow non-invasive diagnostics of living cells and red blood cells by giant Raman spectroscopy as a result of the stage of mixing of blood plasma cells with colloidal solutions of nanoparticles having less effective contact with cells, a longer period of cell immobilization.

Disclosure of invention

The objective and technical result of the present invention are to create a method for the analysis of membrane-bound hemoglobin in red blood cells using giant Raman spectroscopy on nanostructured coatings for the diagnosis of living cells. The use of nanostructured substrates with controlled morphology does not cause erythrocyte hemolysis and allows them to be diagnosed by SEC as a result of more effective contact of nanoparticles (NPs) with living blood plasma cells, eliminating the stage of mixing of red blood cells with colloidal solutions of NPs and reducing the time of cell immobilization.

The problem is solved in that the claimed method for the analysis of membrane-bound hemoglobin in erythrocytes using giant Raman spectroscopy (GCR) is characterized in that nanostructured coatings in the form of ring silver nanostructures having a hierarchical structure are used for research, while the rims of the silver rings consist of interconnected micron-sized porous silver aggregates, on the surface of which rounded silver nanoparticles are located and embedded into the matrix and the size of 2-100 nm.

Also, the problem is solved by the fact that the nanostructured coatings with controlled morphology used in the above analysis method do not cause erythrocyte hemolysis and allow their diagnosis by SEC.

A particular embodiment of the present invention is the aforementioned method, which includes preparing blood plasma cells and applying them to nanostructured coatings, followed by immobilization and removal of the SERS spectra.

A particular embodiment of the present invention is the aforementioned method, according to which the time of immobilization of cells on nanostructured coatings is from 5 to 40 minutes.

Another particular embodiment of the present invention is the aforementioned method, according to which the SEC spectra are obtained using green lasers with a wavelength of 514 or 532 nm.

The invention is illustrated by figures and examples.

Description of drawings

Figure 1. (a) Raman and SQR spectra of erythrocytes, erythrocyte shadows, and isolated hemoglobin obtained under various conditions. Gray color indicates the average deviation from the experimental results, black lines show the averaged spectra. (1) The Raman spectrum of blood diluted 3,000 times; and (2) whole blood applied to a glass slide. (3) The SEC spectrum of a suspension of red blood cells obtained by diluting blood 3,000 times and damaged by a laser beam with a wavelength of 514 nm and with a radiation power 20 times higher than the power used in other SERS experiments. (4) The SEC spectrum of an erythrocyte suspension obtained by diluting blood 5,000 times, obtained by averaging the SEC spectra recorded from various regions of the substrate using a 532 nm laser. (5) A series of SEC spectra of a erythrocyte suspension obtained by dilution of blood 3,000 times, SEC spectra were recorded using a 514 nm laser from the same region on a substrate with a difference of 2 min between measurements. (6, 7) SEC spectra of a erythrocyte suspension obtained by diluting blood 3,000 times, recorded using a 514 nm laser and various substrates with overlapping silver rings visually looking like a “silver mirror”, (b) SEC spectra of isolated hemoglobin and shadows erythrocytes on nanostructured silver substrates (in all cases, the laser wavelength is 514 nm). The SEC spectra of isolated hemoglobin at concentrations of 1 μM (1) and 0.33 nM (2), shadow of erythrocytes with a low content of GB _ms 15 minutes after application to the substrates necessary for the attachment of shadows to the substrate (3). The SQR spectra of the erythrocyte shadow with GB _ms recorded from the same region of the substrate and the time difference between each spectrum of 2 min (4-8). (c) Scheme of the porphyrin complex of iron with characteristic vibrations of bonds indicated by color. (d) A diagram illustrating the proposed locations of red blood cells on a nanostructured silver substrate: 1 - on the wall, 2 - at the point of contact with the wall, 3 - in the area with a high density of silver clusters, 4 - in the area that does not contain silver.

The implementation of the invention

Blood was obtained from the tail vein of male Wistar rats. Dilution of blood 3000 or 5000 times was carried out using Alain physiological solution with the composition: pH = 7.4, 145 mM NaCl, 5 mM KCl, 4 mM KCl, 4 mM Na ₂ HPO ₄ , 1 mM NaH ₂ PO ₄ , 1 mM MgSO ₄ , 1 mM CaCl ₂ .

Example 1

For scanning electron microscopy (SEM) studies and for performing SERS mapping of red blood cells, a whole blood sample was centrifuged (3000 g, 10 min) and the supernatant was removed. A drop of erythrocyte suspension concentrated after centrifugation was applied to coatings with ring silver metal nanostructures (Eugene A. Goodilin et al., Planar SERS nanostructures with stochastic silver ring morphology for biosensor chips, J. Mater. Chem., 2012, 22, 24479-2495 %). The SERS spectra of the samples were obtained using green lasers with a wavelength of 514 or 532 nm.

Example 2

Isolated hemoglobin was isolated using a phosphate buffer (Na ₂ HPO ₄ : NaH ₂ PO ₄ · 2H ₂ O = 4: 1) with low osmolarity and pH = 7.2. Red blood cell hemolysis proceeded with the destruction of the plasma membrane and the release of cytoplasmic hemoglobin into the buffer solution. To clean the cytoplasmic hemoglobin from the remnants of the plasma, the erythrocyte mass was diluted 5-15 times with buffer, centrifuged for 10 min (5000 g), and the obtained supernatant was selected, which was used to record the Raman spectra of hemoglobin on coatings with ring silver metal nanostructures (the method of obtaining example 1). The SERS spectra of the samples were obtained using green lasers with a wavelength of 514 or 532 nm. The hemoglobin concentration in the supernatant, estimated from the absorption spectra, was 10 ^-4 M.

Example 3

To obtain "shadows" of red blood cells (closed vesicles consisting of a plasma membrane of red blood cells with GB _ms ), blood plasma was separated from red blood cells by centrifugation of blood (440 g, 10 min) in phosphate buffer (pH = 7.2) at 4 ° C. The number of red blood cells was counted in the Goryaev’s chamber. Using phosphate buffer, the number of red blood cells was adjusted to 10 ⁷ cells / μl. The resulting erythrocyte suspension was diluted 20 times with ice-cold phosphate buffer (pH = 7.4) and centrifuged at 4 ° C (4000 g, 40 min). The supernatant containing cytoplasmic hemoglobin was separated. The pellet containing erythrocyte shadows was suspended in freshly prepared ice-cold phosphate buffer (pH 7.4) and centrifuged at 4 ° C (4000 rpm, 40 min). The procedure was repeated 5 times. Next, concentration of erythrocyte shadows was carried out in Alain's buffer (pH = 7.4) by centrifuging the sample (10000 g, 30 min). The obtained erythrocyte shadows had a pinkish color due to the presence of membrane-bound hemoglobin attached to the AE1 membrane exchanger. GCR analysis of the obtained “shadows” of erythrocytes was carried out on coatings with ring metal silver nanostructures (the method of preparation in Example 1) using green lasers with a wavelength of 514 or 532 nm.

Example 4

Shadow of erythrocytes with a low content of membrane-bound hemoglobin was obtained by a similar method, replacing a phosphate buffer with pH = 7.2 with a buffer with pH = 8.0. The alkaline medium facilitated the separation of a larger number of membrane-bound hemoglobin molecules from AE1 protein. Unlike the previous ones, shadows without membrane-bound hemoglobin are whitish. For GCR experiments, a suspension with erythrocyte shadows was diluted 1000 times and applied to coatings with ring silver metal nanostructures (the preparation method in Example 1). The SERS spectra of the samples were obtained using green lasers with a wavelength of 514 or 532 nm.

As can be seen from FIG. 1, when blood is diluted, the usual Raman signal disappears: for a blood sample diluted 3,000 times and placed on a glass slide, there is no Raman spectrum (Fig. 1a, spectra 1 and 2). This indicates that the Raman spectrum of highly diluted samples cannot be recorded without a SERS active substrate. It was shown that laser radiation with a wavelength of 514 and 532 nm with an optimally selected power (6 mW per spot with a diameter of 2 mm) allows you to keep red blood cells in a living state. HR analysis of isolated GB and erythrocyte shadow with GB _ms in the presence of nanostructured silver substrates showed that the observed HRG spectra of red blood cells correspond to vibrations of hemoporphyrin bonds in GB _ms (Fig. 1, b). So, the SQR spectra of living red blood cells (Fig. 1, a, spectra 4-7) correspond to the amplified Raman signal GB _ms . In the SEC spectra of erythrocytes on nanostructured coatings with ring structures (Fig. 1a, spectra 4 and 5), the most intense peak is observed at 1370–1375 cm ^–1 , as well as two overlapping peaks in the regions of 1560–1588 and 1620–1630 cm ^-1 arising due to the enhancement of the characteristic Raman peaks of hemoporphyrin GB located at 1550, 1564, 1588 cm ^-1 and 1622, 1640 cm ^-1 , respectively.

Thus, the experimental data confirm that the claimed method allows you to analyze membrane-bound hemoglobin in living red blood cells using giant Raman spectroscopy.

Claims (4)

1. The method of analysis of membrane-bound hemoglobin in red blood cells using giant Raman spectroscopy (GCR), characterized in that the study uses nanostructured coatings in the form of ring silver nanostructures having a hierarchical structure, while the rims of the silver rings consist of porous aggregates communicating with each other micron-sized silver, on the surface of which rounded silver nanoparticles 2-100 nm in size are located and embedded in the matrix. 2. The method according to claim 1, characterized in that the nanostructured coatings with controlled morphology do not cause erythrocyte hemolysis and allow them to be diagnosed by SEC. 3. The method according to claim 1, characterized in that the time of immobilization of cells on nanostructured coatings is from 5 to 40 minutes. 4. The method according to claim 1, characterized in that the SEC spectra are obtained using green lasers with a wavelength of 514 or 532 nm.

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