WO2024080956A1

WO2024080956A1 - A method for obtaining a device used in biomarker detection

Info

Publication number: WO2024080956A1
Application number: PCT/TR2023/051110
Authority: WO
Inventors: Fatih INCI; Yusuf ASLAN; Kutay SAGDIC; Esma DERIN
Original assignee: Bilkent Universitesi Ulusal Nanoteknoloji Arastirma Merkezi
Priority date: 2022-10-11
Filing date: 2023-10-11
Publication date: 2024-04-18

Abstract

The present invention relates to a method (100) for obtaining a portable measuring device which enables to perform detection and determination of biomarkers by means of metamaterial-based plasmonic sensors that are enhanced with fluorescence and wherein distance to the surface is determined.

Description

A METHOD FOR OBTAINING A DEVICE USED IN BIOMARKER DETECTION

Technical Field

The present invention relates to a method for obtaining a portable measuring device which enables to perform detection and determination of biomarkers by means of metamaterial-based plasmonic sensors that are enhanced with fluorescence and wherein distance to the surface is determined.

Background of the Invention

Metamaterials are rationally designed structures which do not exist naturally. These materials are nano structured interfaces containing thin layers of plasmonic or dielectric materials in sub -wavelength thicknesses which manipulate light by means of spatially arranged meta-atoms. These meta-atoms can directly modify light properties such as phase, amplitude and polarisation and thus enhance the photonic behaviour of natural materials. Therefore, metamaterials are used in flat lenses, holograms, invisible skin capes and photonic devices. The light control capability of metamaterials is substantially dependent on the designed nanostructures, particularly their shape and material composition. In addition, metamaterial-based plasmonic surfaces provide various advantages in comparison to classical surface plasmon (SP) platforms: namely, 1) ultra-precise tuning of electromagnetic spectra in order to increase sensitivity, 2) flexibility in production, 3) stability in signal readout, 4) lower radiation attenuation and higher quality factor, and 5) creation of multiple resonances. These materials consist of colloidal, planar or periodically patterned nanostructures. With these structures, localised light reconstruction is ensured at sub-wavelength scales. For instance, metamaterials created by combining photonic and plasmonic colloidal particles have allowed cell imaging and detection of disease biomarker. Unfortunately, during this combination process, nanoparticles lose either their optical or photonic behaviours. Although the optical properties of the metamaterial remain stable when a certain distance is maintained among the particles by using polymeric materials, the applicability of these structures has been limited to cell imaging and labelling. Hyperbolic metamaterials, which are designed in planar forms, display high sensitivity to bio-sensitivity. However, these structures can only be created with complex, laborious, time-consuming and expensive systems including the overlaying of multiple materials with different properties. These systems are monitored by measurements in the near infrared or infrared wavelengths. Thus, they are unlikely to be applicable at the bedside without complex equipment and a specialised person.

In order to overcome these difficulties in metamaterial production, researchers have already utilized surfaces with periodic patterns. Most notably, taking their most important examples from nanoscale patterns in nature, structures with periodic nanopatterns (for example, honeycomb) produced by bio-inspired and microcontact imprinting strategies have solved part of the above-mentioned production problems. Additionally, photonic crystal opals and plasmonic sensors were produced by using biocompatible silk and the presence of biological molecules was detected by changes based on refractive index difference. In the recent times, researchers have succeeded in producing plasmonic surfaces by using periodic nanopatterns included on optical discs. Besides refractive index changes, detection of biological molecule was also performed on these surfaces. Recently, detection of biological molecules (proteins and virus particles) were performed on a Digital Versatile Disc (DVD)-based plasmonic surface. However, metamaterial plasmonic sensors made only with DVD surfaces do not enable to take measurements that are sufficiently sensitive. Therefore, the requirement to increase the sensitivity on the sensor surface is evident. In such cases, gold nanoparticles and molecules with fluorescent structure are used for enhancement of signal and sensitivity on plasmonic surfaces. In particular, fluorophores, quantum dots and nanocrystals have been applied in biomolecular imaging and sensor technologies for a long time. For example, by using photonic materials, Forster Resonance Energy Transfer (FRET) enables the transfer of excitations from donor particles to acceptor particles by means of non-irradiative dipoledipole interactions. FRET strategies are widely used in applications ranging from colour tuning to energy harvesting and sensors. However, even if nanocrystals in larger sizes are used (due to the centre-to-centre distance), FRET efficiency is limited to a length scale of about 10 nm. At longer distances (16-20 nm), the effect of FRET becomes insignificant. Since the FRET effect decreases in the ratio of 1/r⁶ and it is substituted by the Purcell effect (1/r³) (r: separation distance between plasmon and fluorophore). A photoluminescence effect is created upon fluorophores are integrated on plasmonic surfaces and thus, the strength of donoracceptor interactions is increased. However, because these measurements are performed on a small surface area with complex equipment, they are not sensors which can be used in bedside diagnostic tests. In addition, nanopattemed structures are created by transferring the nanostructures on optical discs to polydimethylsiloxane (PDMS) stamp at first and then to polymethylsilsesquioxane (PMSSQ) material by means of micro-contact printing method. It has been found that when fluorescence molecules are placed on randomly occurring nano-slots in nanopatterned structures in order to enhance surface sensitivity, the signal is increased by 69 to 118 times. However, these nano-spaces occur completely randomly during the production of plasmonic surfaces and there is no control and standardisation of this process in any way. Furthermore, these periodic nanostructures cannot be produced in a large area and cannot maintain their nanopattern dimensions on the PDMS stamp after a certain number of uses. All these demonstrate that the metamaterial techniques applied today have fundamental problems in production and method standardisation and subsequently technological gaps and clearly indicate the need for a portable biomarker detection device suitable for use at the bedside. The International patent document no. W02009021964, an application included in the state of the art, discloses a series of optical elements configured to ensure enhancement of optical output by means of a combination of plasmonic effects, including surface plasmon resonance and surface plasmon-coupled emission. Such optical element can be geometrically configured to preferentially distinguish the angular distribution of light emitted to the optical element, such as the ones provided by configurations optimized to utilize supercritical angle fluorescence phenomena. In one embodiment, incorporation of a plasmonic enhancement phenomenon into a high-efficiency collector of fluorescence generated by a biochip surface provides high-efficiency optical elements having application in the detection of small amounts of analyte. The optical element provided is a sensitive device used particularly in biomedical diagnostics application.

Summary of the Invention

An objective of the present invention is to realize a method for obtaining a portable measuring device which enables to perform detection and determination of biomarkers by means of metamaterial-based plasmonic sensors that are enhanced with fluorescence and wherein distance to the surface is determined.

Another objective of the present invention is to realize a portable measuring device which is suitable for use both in the clinic and at the bedside.

Another objective of the present invention is to realize a method for obtaining a metamaterial by using periodic nanopatterns already present on optical discs which remain on shelves, are unused and appear as idle.

Another objective of the present invention is to realize a method for obtaining a measuring device by combining plasmonic, photonic and microfluidic structures. Another objective of the present invention is to realize a measuring device which is capable of recognizing a plurality of biomarkers.

Another objective of the present invention is to realize a method for obtaining a device which is low cost, easy-to-use, capable of performing measurement with high precision and does not require specialised personnel for measurement.

Detailed Description of the Invention

“A Method for Obtaining a Device Used in Biomarker Detection” realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

Figure l is a flow chart of the inventive method.

Figure 2 is projection drawings of the invention, a) DVD plastic mould and a plurality of metal coatings (Titanium: 10 nm; Silver: 30 nm; Gold: 15 nm) b) Short-range modification obtained with 3-MNHS (-4 nm), c) Medium-range modification obtained with SH-PEG 600-biotin (-6-10 nm), d) Long-range modification obtained with SH-PEG 2000-biotin (-16- 20 nm).

Figure 3. AFM and SEM images of a) DVD plastic mould and b) metamaterial plasmonic sensor formed by a plurality of metal coatings (Titanium: 10 nm; Silver: 30 nm; Gold: 15 nm) on this mould.

Figure 4. Surface chemistry applications, (a-b) short-range (-4 nm) modifications with 3-MNHS, (c-d) medium-range (-6-10 nm) modifications with SH-PEG 600-biotin, (e-f) long-range (-16-20 nm) modifications with SH-PEG 2000-biotin are shown.

Figure 5. Avidin-FITC application to (a) Avidin-FITC and avidin molecules (b) short, (c) medium and (d) long-distance modifications. Figure 6. Binding of avidin and avidin-FITC proteins on (a-b) on short- range modification, (c-d) on medium-range modification and (e-f) on long- range modification. Comparison of all modifications by normalisation (between 0-1) of (g-h) Violin-box plot and data are shown. Data were analysed by performing non-parametric Kruskal-Wallis statistical analysis and statistical difference is indicated as * p<0.05 and ** p<0.01.

Figure 7. Binding of Avidin-FITC proteins in artificial urine on the (a-b) sensor surface modified with SH-PEG 600-biotin. (c) Changes in the plasmonic resonance value of different glycerol solutions (l%-70%) by final measurement analysis, (d) Illustration of the effect of glycerol solutions of different concentration and refractive index on the sensor with and without modification.

Figure 8. Fluorescence microscopy imaging, (a) Images obtained during blank, avidin-FITC and washing, respectively, (b) Comparison of green light intensity resulting from analysis and (c) normalised data are shown.

The components illustrated in the figures are individually numbered, where the numbers refer to the following:

100. Method

The inventive method (100) for obtaining a portable measuring device for detecting disease-causing biomarkers included in biological fluids such as blood, serum or urine both in the clinic and at the bedside by using a fluorescence- enhanced metamaterial plasmonic sensor, comprises the steps of performing chemical etching by revealing the nanoperiodic structure on the optical disc (DVD) surface (101); obtaining a metamaterial plasmonic sensor by coating the etched surface

(102); creating a microchannel on the metamaterial plasmonic sensor surface

(103); and obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and long-distance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104).

In the step of performing chemical etching by revealing the nanoperiodic structure on the optical disc (DVD) surface (101) of the inventive method (100); plastics which are located on the optical disc surface and unwanted are removed from the surface by means of a scalpel (bistoury). Then, the surface is cleaned with an ethanol-methanol mixture prepared at a ratio of 1 : 1 by volume and paint and unwanted layers are cleansed from the surface. Chemical etching is carried out by applying a mixture of acetone-isopropanol prepared at a ratio of 1 :4 by volume to the cleaned surface for 30-120 seconds until the desired periodic structure is obtained.

In the step of obtaining a metamaterial plasmonic sensor by coating the etched surface (102) of the inventive method (100); a metamaterial plasmonic sensor is obtained by coating the etched surfaces with 5-15 nm titanium, 25-50 nm silver and 5-20 nm gold, respectively, by using an electron-beam evaporation method.

In the step of creating a microchannel on the metamaterial plasmonic sensor surface (103) of the inventive method (100); microchannels of desired dimensions are created on the surface by integrating a microfluidic chip on the surface of the metamaterial sensor. In one embodiment of the invention, the created microchannels have dimensions of (4-10 mm) x (10-40 mm) x (25-250 pm); width x length x height. In one embodiment of the invention, the plasmonic sensor has a dimension of (10-20 mm x 15-50 mm).

In the step of obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and long-distance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104) of the inventive method (100); fluorescent materials are integrated on the sensor surface by means of biomolecules in order to enhance the sensitivity of the obtained metamaterial plasmonic sensor to biomarkers. In one embodiment of the invention, the fluorescent material used is fluorescein isothionocyanate (FITC). Avidin-biotin interaction is used to bind the fluorescent material to biomolecules, and avidin-FITC molecules are selected for this interaction. The fluorescent material is applied to the metamaterial plasmonic sensor surface with a distance of 6-10 nm.

The portable measurement device obtained by means of the inventive method (100) enables to measure disease-causing biomarkers in biological fluids such as blood, serum or urine with low cost and high sensitivity both in the clinic and at the bedside.

The experimental process and characterisation studies carried out to achieve the measurement device which performs biomarker determination by means of the inventive method (100) are as follows: The nanoperiodic structure included on the surface of optical discs (DVD) is used for sensor production. Firstly, in order to enable the periodic structure on the DVD surfaces to provide the plasmonic effect, a chemical etching process was applied with organic solvents and the surfaces were made ready for metal coating in the next stage. For this process, unwanted plastics included on the DVD surface were simply removed from the surface by using a scalpel. Subsequently, these surfaces cleaned with a mixture of ethanolmethanol (1 : 1, volume: volume) were cleansed from the related paint and unwanted layers. As stated in the literature, an etching process was carried out by applying an acetone-isopropanol mixture in a ratio of 1 :4 (volume: volume) for 60 seconds. These surfaces were coated with titanium (10 nm), silver (30 nm) and gold (15 nm) by means of electron-beam evaporation method and metamaterial plasmonic sensors were obtained.

Then, scanning electron microscopy (SEM) was used to observe the continuity of the nanoperiodic area in a larger area. For this process, DVD surfaces being subjected to chemical etching for 60 seconds were pretreated. The surfaces were washed with ethanol at first and sonicated for 5 minutes. Thereby, impurities included on the surface were removed from the environment. Since DVD surfaces have a polycarbonate structure, these surfaces must be coated with a conductive material so as to be visualised in a SEM device. Otherwise, the negative charges accumulating on the surface prevent the characterization method from being carried out correctly. Therefore, the surfaces were coated with a gold-palladium (Au-Pd) mixture by means of sputter coating method. Precision Etching and Coating System (PECS) was used for the coating process. During the process, the device parameters were adjusted so that the coating density was 19.32 g/cm³ and the acoustic resistance was 23.20 x 105 gm/cm²s (acoustic impedance). The film thickness created as a result of the coating process is approximately 10 nm and it does not cause any damage to the nanoperiodic structure. As a result of the SEM characterisation, it was observed that the nanoperiodic structures exhibited continuity in a large area and a curved progression partly. In addition, when these DVD surfaces were analysed at a smaller size at AFM, it was calculated that they had a period of -740 nm (Figure 3a). Similar procedures were applied for the metal-coated metamaterial plasmonic sensor (except for the gold-palladium coating) as well. Here, it was observed that similar structures continued in the wide (SEM) and narrow (AFM) areas following the coating and that the surface period was even more uniform (Figure 3b).

Thereafter, a microchannel (4.5 mm x 10.2 mm x 50 pm; width x length x height) was created on the metamaterial plasmonic sensor (15 mm x 15 mm) by integrating a microfluidic chip for the application of biomolecules. In order to take the sensitivity of the produced plasmonic metamaterial to biomarkers one step further, it was planned to integrate the fluorescence materials on the surface by means of biomolecules. Thus, the effect of special conditions that may occur as a result of changing the distance of the fluorescence molecules to the surface on the plasmonic signal was aimed to be investigated. In the literature, the possible interactions of the fluorescence molecules with the plasmonic surfaces were investigated and as a result, two different types of optical states were predicted. The first case is related to the non-radiative heat energy transferd of the fluorescence molecules established with the nanostructures and this effect is called as FRET. The FRET effect occurs when the distance of the fluorescence molecules to the surface is less than about 10 nm. Whereas the second case is based on the fact that the fluorescence molecule moves away from the surface by more than about 10 nm, gets free from the FRET effect and the resulting emission signal is enhanced by the increasing electromagnetic field. This effect is referred to as the Purcell effect in the literature. Considering the two optical effects mentioned above, fluorescein isothionocyanate (FITC) was chosen as the fluorescent material in this study. In this choice, the fact that the emission wavelength and plasmonic absorption wavelengths are included in similar ranges in the spectrum was effective. The binding of the fluorescence molecules to the biomolecules was desired to utilize the avidin-biotin interaction and avidin-FITC molecules were used. The distance of FITC to the surface was determined in three steps. (1) Since it is the avidin molecules which provide the actual distance between the sensor and the fluorophores in the short-range modification, the size of the avidin was the determining factor and it was reported as ~4 nm in the literature. Binding to the gold part of the metamaterial sensor with the thiol groups was realized by using the molecule 3-mercaptopropanil-N-hydroxysuccinimidyl propionate (10 mM) mediator here and the succinimide groups were used to bind the avidin-FITC molecules. In this configuration, FRET (~4 nm) interaction was observed. (2) By applying a mid-distance modification, SH-PEG 600-biotin (molecular weight: 600 Da) (10 mM) was used to increase the distance between the sensor and the fluorophores. The thiol functional group located at the end of this polymeric molecule interacts with the gold metamaterial surface and presents the biotin moiety to the surface. Then, avidinfluorophores were introduced into the system at different concentrations and a layer caoted with a fluorophorewas created at a certain height from the sensor surface. As stated in the literature, the length of the PEG molecule is related to the amount of repeated ethylene glycol it has. The number of ethylene glycols (n) is determined by the formula of PEG molecular weight (18.02 + (44.05 x n). For example, 13 sub-units (ethylene glycol) are included in PEG 600. Depending on the orientation of the bonds, the length of each sub-unit segment varies between 0.278 nm-0.358 nm. The sizes of the complex -which is created by the biotin integrated with the intermediary molecule avidin used- also varies between 4.2 nm-5.8 nm (Weber et al., 1992; Weber et al., 1989). Thus, SH-PEG 600-biotin is approximately 7.8 nm-10.4 nm in size (the thiol size was not taken into account because it is at the angstrom level). With this modification, the transition between FRET-Purcell was monitored by setting the distance between the sensor and the fluorophores to 6-10 nm. (3) By applying long-distance modification, it was aimed to see more Purcell effect and the modification strategy mentioned in (2) was achieved with SH-PEG 2000-biotin (molecular weight: 2000 Da) (10 mM) which is a longer polymeric molecule. As calculated above, the height provided to the fluorophore by the intermediate molecule here is approximately 16.7 nm-21.9 nm. All surface chemistry strategies are shown in the Figures 4 and 5.

As it is common at the beginning of short, medium and long-distance modification experiments, the background signal (air) was received from the empty chip in order to create a reference line at the beginning of the measurement. Then, distilled water was sent to the microfluidic chip by means of hoses via a syringe pump at a constant speed (5 pL/min) for approximately 350 s in order to obtain a stable signal value. Avidin protein (control) (100 nM) or avidin-FITC was sent to the sensor surface equilibrated with distilled water for 3200 seconds. When the signal changed from a rising trend to a balanced approach, a washing process was applied with water again for 300 seconds in order to remove proteins that could not adhere to the chip surface (Figure 6a-f). In short-, medium- and long-range modification, the binding of avidin protein caused an average plasmonic resonance shift of 1.23 nm, 0.36 nm and 0.22 nm, respectively; whereas in avidin-FITC experiments, these average values were calculated as 1.04 nm, 1.89 nm and 0.83 nm (Figure 6g). Subsequently, when we normalized the data (0-1), the values were calculated as 0.68, 0.20 and 0.17, respectively in the event that avidin was bound to the short, medium and long-modified surfaces; whereas these values were 0.58, 0.90 and 0.47 in the avidin-FITC experiments (Figure 6h). Additionally, when we compared each of three experimental setup with their own control groups, it was observed that the same concentration of avidin-FITC molecule reduced the plasmonic signal (in the short-range modification) in the presence of a modification near the surface; whereas avidin- FITC molecule at a distance from the sensor surface increased the plasmonic effect (in the middle and in long-range modifications). As a result, a signal loss of approximately 15% in the short distance (FRET effect) was observed; whereas a signal enhancement of approximately 4.5 and 2.8 times (Purcell) was observed in the medium and long distance modifications, respectively. For application purposes in light of these results, it is shown that if the surface chemistry is to be carried out close to the sensor (~4 nm), the fluorescent molecule will reduce the signal, and if a fluorescent molecule is to be used, mid-range surface modification will be more efficient.

Biological fluids (e.g., blood, serum or urine) vary greatly in terms of both their density and content (ionic strength, ionic charge and protein/sugar content) compared to water and/or phosphate buffer solution (PBS). In order to understand the performance of the sensor we developed and its specificity for disease markers, test liquids were at first by changing the water medium so as to contain 100 nM avidin-FITC in artificial urine samples and the same system was tested on SH-PEG 600-biotin-containing surfaces. In this experimental setup, a plasmonic resonance shift of approximately 2.54 nm was observed (Figure 7a-b). As controls, two different experimental setups were organised: (1) artificial urine, which does not comprise avidin-FITC and (2) 100 nM bovine serum albumin (BSA) were introduced into the system (Figure 7b). In these two controls, the initial data obtained with water increased for a certain period of time after the introduction of artificial urine with a different refractive index and then stabilised (Figure 7b). Upon washing was performed with water, the signal decreased to the starting point (the same level as the water data) and did not lead to any plasmonic resonance shift on the surface (Figure 7b). Furthermore, by applying different glycerol concentrations (l%-70%) on SH-PEG 600-biotin modified surfaces (after Avidin FITC binding), the sensitivity of the developed sensor to refractive index changes was investigated and compared with the sensor without any modification (Figure 7c-d). In the observed results, the sensors with and without modification exhibited similar results in refractive index changes (Figure 7c-d). When we analysed the results in the Figures 6 and 7 together, it was observed that the avidin-FITC molecule bound to the surface caused more plasmonic resonance shift when placed at a certain distance from the surface and exhibited similar results in terms of refractive index with unmodified sensors, suggesting that this structure increases the binding sensitivity.

In the next step, the measurement of sensor sensitivity performance was performed. The refractive index sensitivity and figure-of-merit (FOM) of the sensor were calculated by analysing the sensor signal changes based on the refractive index changes in liquids. In the glycerol experiments shown in the Figure 7 (l%-70%, volume: volume), the magnitude of the observed shifts in the resonance wavelength values was divided by the change in refractive index and then the refractive index sensitivity was calculated as 338.5 nm/refractive index. In addition, FOM was calculated by dividing the refractive index by the full width at half maximum (FWHM). The values used in the sensitivity performance measurement are summarised in Table 1.

Table 1. Sensor performance parameters. Glycerol concentrations, refractive index (RIU), full width at half maximum (FWHM) and figure of merit (FOM).

In addition, images were taken from the blank surface before avidin-FITC protein binding (i), the protein-bound surface (ii) and the surface formed after washing (iii), by means of vertical light microscopy on surfaces with mid-range surface modification (SH-PEG 600-biotin) (Figure 8a). By using the open-source application Fiji-ImageJ (NIH), green light intensity analysis was performed on the images taken for the 3 cases mentioned above (Figure 8b). Green colour intensity was found to be highest on the avidin-FITC protein bound surface and lowest on the blank surface. When the green light intensity data were normalised (0-1), it was calculated as 0.15, 0.84 and 0.51, respectively (Figure 8c). It was observed that the avidin-FITC protein bound to the surface remained approximately 60% on the surface after washing and maintained its brightness.

Within these basic concepts; it is possible to develop various embodiments of the inventive “Method (100) for Obtaining a Device Used in Biomarker Detection”; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

1. A method (100) for obtaining a portable measuring device for detecting disease-causing biomarkers included in biological fluids such as blood, serum or urine both in the clinic and at the bedside by using a fluorescence- enhanced metamaterial plasmonic sensor; characterized by steps of performing chemical etching by revealing the nanoperiodic structure on the optical disc (DVD) surface (101); obtaining a metamaterial plasmonic sensor by coating the etched surface (102); creating a microchannel on the metamaterial plasmonic sensor surface (103); and obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and longdistance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104).

2. A method (100) according to Claim 1; characterized in that in the step of performing chemical etching by revealing the nanoperiodic structure on the optical disc (DVD) surface (101); plastics which are located on the optical disc surface and unwanted are removed from the surface by means of a scalpel (bistoury).

3. A method (100) according to Claim 2; characterized in that in the step of performing chemical etching by revealing the nanoperiodic structure on the optical disc (DVD) surface (101); the surface is cleaned with an ethanolmethanol mixture prepared at a ratio of 1 :1 by volume and paint and unwanted layers are removed from the surface.

4. A method (100) according to Claim 3; characterized in that in the step of performing chemical etching by revealing the nanoperiodic structure on the optical disc (DVD) surface (101); chemical etching is carried out by applying a mixture of acetone-isopropanol prepared at a ratio of 1 :4 by volume to the cleaned surface for 30-120 seconds until the desired periodic structure is obtained. A method (100) according to any of the preceding claims; characterized in that in the step of obtaining a metamaterial plasmonic sensor by coating the etched surface (102); a metamaterial plasmonic sensor is obtained by coating the etched surfaces with 5-15 nm titanium, 25-50 nm silver and 5- 20 nm gold, respectively, by using an electron-beam evaporation method. A method (100) according to Claim 5; characterized in that in the step of creating a microchannel on the metamaterial plasmonic sensor surface (103); microchannels of desired dimensions are created on the surface by integrating a microfluidic chip on the surface of the metamaterial sensor. A method (100) according to Claim 6; characterized in that in the step of creating a microchannel on the metamaterial plasmonic sensor surface (103); the created microchannels have dimensions of (4-10 mm) x (10-40 mm) x (25-250 pm); width x length x height. A method (100) according to Claim 6; characterized in that in the step of creating a microchannel on the metamaterial plasmonic sensor surface (103); the plasmonic sensor has a dimension of (10-20 mm x 15-50 mm). A method (100) according to any of the preceding claims; characterized in that in the step of obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and longdistance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104); fluorescent materials are integrated on the sensor surface by means of biomolecules in order to enhance the sensitivity of the obtained metamaterial plasmonic sensor to biomarkers. A method (100) according to Claim 9; characterized in that in the step of obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and long-distance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104); the fluorescent material used is fluorescein isothionocyanate (FITC). A method (100) according to Claim 10; characterized in that in the step of obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and long-distance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104); avidin-biotin interaction is used to bind the fluorescent material to biomolecules, and avidin-FITC molecules are selected for this interaction. A method (100) according to Claim 11; characterized in that in the step of putting a liquid used for creating a colloidal quantum well (K) film into a container (101); obtaining a biomarker measurement device by integrating fluorescence material upon applying short, medium and long-distance modifications to the metamaterial plasmonic sensor on the surface of which microchannel is created (104); the fluorescent material is applied to the metamaterial plasmonic sensor surface with a distance of 6-10 nm.