Novel self-assembled organic nano sensor for albumin detection

Abstract

The invention describes synthesis of a novel self-assembled organic nano sensor. The self-assembled organic nano sensor takes naphthalene sulfonate as a framework, and the size distribution is 40-2000 nanometers. The autofluorescence of the self-assembled organic nano sensor is very low, but the fluorescence activity is very high in the presence of albumin, so that the signal-to-noise ratio is very high. The generated fluorescence is in direct proportion to the concentration of albumin, and can be used for quantifying albumin in a sample. The self-assembled organic nano sensor can be used for linearly quantifying the albumin concentration in a range of 0.1 mg / L-3500mg / L. After the self-assembled organic nano sensor is introduced into a sample containing albumin for 5-10 seconds, the nano sensor can emit fluorescence. Through interaction with the self-assembled organic nano sensor, albumin in an aqueous solution shows stability of 20 days, and the stability is kept between-80 DEG C and + 95 DEG C.

Description

TECHNICAL FIELD

[0001] The present invention relates to a novel method of synthesis of self-assembled organic nanosensor and a method of detecting albumin using the nanosensor. More specifically, the present invention describes a method of preparation of self-assembled organic nanosensor which has very low autofluorescence and emits stable high fluorescence upon photoexcitation in the presence of albumin. The self-assembled organic nanosensor can be used for qualitative and quantitative estimation of albumin in solution and solid supports. BACKGROUND

[0002] Albumin is a very important protein, accounting for 54% of egg protein, 22% of milk protein, 36% of whey protein, and 50-60% of human serum protein. Apart from nutritional value, albumin has wide industrial and research applications. Egg white protein is an important ingredient in many foods, including pet foods, due to its heat coagulation and binding properties. Bovine serum albumin is used in vaccine production and has great value in cell culture related work, including efficient washing of cells and cryopreservation. Human serum albumin has been used as a biomarker for detecting various physiological conditions and health monitoring. Qualitative and quantitative estimation is very important for its industrial and research applications and quality control. According to Hosen et al. 2013, albumin in fake eggs made with sodium alginate can be identified on-site by rapid qualitative analysis. According to Smuts et al. 2019, the serum albumin concentration of donor cows determines the developmental capacity of oocytes, and if quantitative determination can be performed on-site, it will be helpful. Serum albumin is used for research on higher specificity and better pharmacokinetic performance of various drugs. Albumin is useful for detecting kidney health using urine samples or detecting dehydration using high concentration serum samples.

[0003] Albumin can be qualitatively and quantitatively detected by various sensors. Some of the existing technologies in terms of albumin detection mechanisms are as follows.

[0004] Colorimetric sensors, organic dyes undergo color indicating reactions with albumin. Folin-Ciocalteu sensor (Lowry method (GB1596914A)) interacts with copper ions and the side chains of tyrosine, tryptophan, and cysteine, producing a blue-green color that can be detected between 650 nm and 750 nm. However, due to slow color development and relatively fast color fading, reasonable precise reaction time and temperature are required. This can be a problem when analyzing many samples and on-site detection.

[0005] The Coomassie Brilliant Blue dye used as a sensor in the Bradford method (US4023933A) binds to positively charged proteins. When the dye is in solution, it is red, with an absorption wavelength of 465 nm, but when it binds to basic amino acids in the protein, it turns blue, with an absorption wavelength of 595 nm. The main disadvantage of this method is that it is very dirty, staining clothes and skin, among others.

[0006] Bromocresol green is an anionic sensor (BCG method (US3873272A)) that interacts with proteins at acidic pH, changing the color of the indicator from yellow-green to green-blue, thus changing the absorption wavelength of the complex. However, this method is very sensitive to pH conditions, and its changes can lead to false positives or false negatives.

[0007] Bicinchoninic acid is used as a sensor (BCA method (DE602004007769T2)). The principle of this method is that proteins can reduce Cu 2+ to Cu 1+ in alkaline solution (biuret reaction) and cause the formation of bicinchoninic acid, which is purple.

[0008] The immunochromatographic method is a qualitative method, where the result is visually negative or positive. This measurement also takes a long time, usually 10-15 minutes, until the liquid sample has completed its movement along the chromatographic strip.

[0009] The immunoturbidimetric method is an antibody-based method, where albumin-specific antibodies interact with the albumin in the sample to form a complex. The correct balance of albumin and antibodies leads to the formation of a complex, which can be tracked by a spectrophotometer due to the occurrence of flocculation and an increase in absorbance. This method is fast, but requires a laboratory setup to perform the test. In addition, it is more suitable for cases of low and narrow range of biological concentrations, and cannot measure cases of high biological concentrations.

[0010] Fluorescent sensors

[0011] Protein biosensors based on fluorescent organic materials have attracted attention due to their functionality, sensitivity, selectivity, and speed. Anionic cyanine dyes (US5182214A) have high sensitivity to HSA, with a linear response in the range of 0-40 mg / L, with an emission wavelength of 640 nm. One of the problems often encountered with traditional fluorophores is aggregation-induced quenching (ACQ), which occurs due to the presence of π-π stacking interactions in planar fluorophores. When the fluorophore is dispersed in an aqueous medium or binds to a protein in a buffered solution, the molecules tend to aggregate, quenching the fluorescence and greatly limiting its effective range as a biological probe.

[0012] However, there is another class of fluorescent organic materials that have the property of aggregation-induced emission (AIE). US20130177991A1 describes sulfonated group functionalized tetraphenyl ethylenes that can evaluate protein in urine with high sensitivity based on AIE. It further describes that AIE-active species in monomeric form emit weakly in PBS buffer, but emit strongly when aggregates are formed. In PBS buffer, its solution emits weakly at 390 nm wavelength, while in the presence of HSA, the AIE compound emits strongly at 475 nm wavelength.

[0013] Nanoparticle-based sensor

[0014] Huang et al. in 2015 described a nanosensor based on metal nanoparticles, such as gold nanoparticle-based sensors. The method is based on the change in surface plasmon response after adsorption of albumin on the surface of gold nanoparticle sensors. Lai et al. in 2016 used gold nanoparticle sensors that appear red in the presence of albumin and blue in the absence of albumin aggregates.

[0015] Therefore, the existing albumin detection methods described in the preceding paragraphs have some drawbacks. The chromogenic dyes have low specificity for albumin. In addition, it is a multi-step reaction that requires laboratory configuration to perform the detection, and therefore is not suitable for on-site, field, or instant detection. The immunoresponsive probes have high specificity, but the detection range is narrow. The detection requires skilled personnel and laboratory equipment. In addition, they can only identify the immunoresponsive part of albumin in the sample, and cannot capture the non-immunoresponsive albumin, resulting in insufficient representation of the total content of albumin in the sample. The fluorescent probes are promising, but the aggregation-induced quenching phenomenon has been reported, and therefore are not suitable for industrial applications.

[0016] The aggregation-induced emission class of fluorescent sensors has great prospects, but their synthesis process is complex and expensive. Therefore, there is an urgent need for a sensor that has a wide range of albumin detection, is easy to detect, requires minimal laboratory equipment and facilities, is easy to synthesize and low cost, has high stability, and is suitable for a wide range of industrial, medical and laboratory applications.

[0017] Nanosensors are nanoscale chemical or mechanical sensors of sub-micron size that can be used to detect the presence of chemical substances and nanoparticles, or to monitor physical parameters such as temperature. They are widely used in medical diagnostics, food and water quality testing, and other chemical fields. Self-assembly of nanostructures is the process by which atoms, molecules, or nanoscale building blocks spontaneously organize into ordered structures or patterns with nanoscale features. It is the most promising low-cost, high-throughput practical nanofabrication method.

[0018] The present invention describes a method for the synthesis of a novel self-assembled organic nanosensor with naphthalene sulfonate as the backbone, which has a wide detection range (0.1-3500 mg / L), high specificity for albumin, low cost, stability, etc., and can detect whether the sample contains albumin without any experimental equipment.

[0019] Therefore, to the best of our knowledge, none of the above prior art attempts alone or collectively suggest or disclose the proposed system and embodiments as set forth and disclosed herein.

[0020] Object of the invention

[0021] The object of the present invention is to synthesize a new self-assembled organic nanosensor with low autofluorescence using the fluorescent monomer backbone of naphthalene sulfonate.

[0022] Another object of the present invention is to provide albumin detection performed by the self-assembled organic nanosensor with high signal-to-noise ratio.

[0023] Still another object of the present invention is to develop a kit that can measure albumin in environmental, biological or synthetic samples in solid or solution form in the field or in situ or instant measurement environment for detection and diagnosis. SUMMARY

[0024] The present invention describes the synthesis of a novel self-assembled organic nanosensor. The self-assembled organic nanosensor has a naphthalene sulfonate backbone and a size distribution of 220-360 nm. The self-assembled organic nanosensor has very low autofluorescence, but in the presence of albumin, it exhibits high fluorescence activity with a high signal-to-noise ratio. The fluorescence produced is directly proportional to the concentration of albumin and can be used to quantify albumin in samples. The self-assembled organic nanosensor can linearly quantify albumin concentrations in the range of 0.1 mg / L-3500 mg / L. Without any device, fluorescence can be observed by visual observation, and fluorescence spectrophotometry or fluorescence imaging devices can be used for qualitative and quantitative analysis. The self-assembled organic nanosensor emits fluorescence within 5 to 10 seconds when introduced into a sample containing albumin. Interaction with the self-assembled organic nanosensor can stabilize albumin in aqueous solution for 20 days. The self-assembled organic nanosensor is stable between -80 degrees Celsius and +95 degrees Celsius. BRIEF DESCRIPTION OF DRAWINGS

[0025] The system and method of the present invention can be fully understood with reference to the following figures.

[0026] Figure 1: Transmission electron microscopy images. 1-A TEM images showing self-assembled organic nanosensors of different sizes from 40 nm to 800 nm; 1-B TEM images showing self-assembled organic nanosensors of different sizes from 500 nm to 2000 nm.

[0027] Figure 2: Dynamic light scattering to characterize the size of monomer and self- assembled organic nanosensor. Particle size distribution histogram of 2-A self- assembled organic nanosensor with a major peak at 229.8 nm. Autocorrelation function plot of self- assembled organic nanosensor (2-B), monomer (2-C), self-assembled organic nanosensor with albumin (2-D), and monomer with albumin (2-E).

[0028] Figure 3: Model of the interaction of albumin with self-assembled organic nanosensor and monomeric scaffold.

[0029] Figure 4: Absorption spectra of monomer and self-assembled organic nanosensor.

[0030] Figure 5: Excitation (5-A) and emission (5-B) fluorescence spectra of monomer and self-assembled organic nanosensor.

[0031] Figure 6: Fluorescence emission spectra of self-assembled organic nanosensor alone and with albumin.

[0032] Figure 7: Concentration plot of self-assembled organic nanosensor with albumin.

[0033] Figure 8: Specificity data of self-assembled organic nanosensor using albumin and non-albumin proteins. Relative fluorescence intensity output of self-assembled organic nanosensor interaction with albumin (human serum albumin (HSA), bovine serum albumin (BSA), lactalbumin, ovalbumin) and non-albumin class proteins (lysozyme, trypsin).

[0034] Figure 9: Stability plot of albumin alone and self-assembled organic nanosensor over 20 days.

[0035] Figure 10: Determination of unknown concentration of albumin in a bovine urine sample using self-assembled organic nanosensor kit. 10-A is a graphical representation of albumin content in a bovine urine sample detected in solution, 10-B shows qualitative detection of bovine serum albumin by interaction with self-assembled organic nanosensor on a solid matrix. Left panel is nitrocellulose paper impregnated with self-assembled organic nanosensor only by slot blot, right panel is nitrocellulose paper impregnated with self-assembled organic nanosensor treated with bovine milk sample only on a UV transilluminator. DETAILED DESCRIPTION

[0036] The following is a detailed description of embodiments of the disclosure described in the accompanying drawings. The detailed description is sufficient to clearly convey the disclosure. The large amount of details provided is intended to encompass all modifications, equivalents, and alternatives that fall within the scope of the disclosure as defined by the appended claims.

[0037] The accompanying drawings incorporated herein and forming a part of the specification, illustrate an embodiment of the present application and together with the description serve to explain the principles of the application.

[0038] In the specification of this document and in the subsequent claims, the meaning of "a", "an", and "the" includes the plural, unless the context clearly dictates otherwise.

[0039] The present invention provides a method for synthesizing self-assembled organic nanosensors. Nanosensors are sub-micron sized nanoscale chemical or mechanical sensors that can be used to detect the presence of chemical substances and nanoparticles, or to monitor physical parameters such as temperature. In the present invention, the nanosensor is a nanoscale chemical sensor. The sensor can be widely used in medical diagnostics, food and water quality testing, and other chemical fields. The nanosensor is synthesized by creating a microenvironment that promotes self-assembly. Self-assembly of nanostructures is the process by which atoms, molecules, or nanoscale building blocks spontaneously organize into ordered structures or patterns with nanoscale features. It is the most promising low-cost, high-throughput practical nanofabrication method. In the present invention, the monomer backbone used has the following chemical properties:

[0040]

[0041] where R can be but is not limited to hydrogen, sodium, carbon, chlorine, fluorine, sulfur, hydroxyl, amine, methyl, oxymethyl, acetyl, acetamide, phenyl, nitoxide.

[0042] In a preferred embodiment, the synthesis process comprises two steps. In the first step, the monomer backbone is dissolved in an alkaline solution, where the concentration of the naphthalenesulfonate monomer backbone can range from 100 millimolar (mM) to 5 molar (M) solution. The pH range of the alkaline solution can be between pH 8-12, where the alkaline solution can be but is not limited to barium hydroxide, sodium hydroxide, ammonium hydroxide, calcium hydroxide, lithium hydroxide, and potassium hydroxide, with a molar concentration of 250 millimolar (mM) to 4 molar (M) sodium bicarbonate. The solution is incubated for 1 hour to 96 hours to allow it to stabilize. The second step is to mix the alkaline solution of the monomer backbone with an aqueous buffer solution with a pH range of 3-5, where the molar concentration of the buffer solution can range from 50 millimolar to 2.5 molar, and the buffer solution can be but is not limited to citrate, formate, ascorbate, phosphate, tris, carbonate. The ratio of the alkaline monomer backbone solution to the buffer solution can range from 0.001 to 10.

[0043] The solution is placed in a closed glass or plastic container and incubated in the dark at 27 to 57 degrees Celsius for 12 to 86 hours. Large self-assembled superstructures are visually observed at the bottom of the container, which are uniformly dispersed after shaking. Then, centrifuge at 10,000 rpm for 10 minutes to collect the precipitate of the self-assembled organic nanosensors. Discard the supernatant and resuspend the self-assembled organic nanosensors in deionized water.

[0044] In another embodiment, the synthesis process comprises dissolving naphthalenesulfonate monomer backbone in an organic solvent, wherein the concentration of naphthalenesulfonate monomer backbone can range from 100 millimolar (mM) to 5 molar (M) solution. The organic solvent can be, but not limited to, dimethylformamide, methanol, tetrahydrofuran and similar solvents, with a concentration range of 10-100%. The solution is incubated for 1 hour to 96 hours to allow it to stabilize. The second step is to mix the monomer backbone in the organic solvent with a buffered aqueous solution with a pH value ranging from 3-5, wherein the molar concentration of the buffered solution can range from 50 millimolar to 2.5 molar, the buffered solution can be, but not limited to, citrate, formate, ascorbate, phosphate, tris-hydroxymethyl aminomethane, carbonate. The ratio of the basic naphthalenesulfonate monomer backbone solution to the buffered aqueous solution can range from 0.001 to 10. The solution is incubated in a closed glass or plastic container in a dark environment at 27 to 57 degrees Celsius for 12 to 86 hours. Large self-assembled superstructures are visible to the naked eye at the bottom of the container, which will be uniformly dispersed after shaking. Then, centrifuge at 10,000 rpm for 10 minutes to collect the precipitate of self-assembled organic nanosensors. Discard the supernatant and resuspend the self-assembled organic nanosensors in deionized water.

[0045] Figure 1 demonstrates the transmission electron microscopy (TEM) images of self-assembled organic nanosensors, wherein the smallest unit of the self-assembled organic nanosensors is about 40 nanometers (nm), and the larger size range of the self-assembled organic nanosensors is about 2000 nanometers. The TEM images of Figure 1-A show self-assembled organic nanosensors of different sizes from 40 nm to 800 nm; the TEM images of Figure 1-B show self-assembled organic nanosensors of different sizes from 500 nm to 2000 nm. The mixture of various preparations of self-assembled organic nanosensors in the electron micrograph grid, with the following preparation conditions: 250 mM to 4 M, pH 8-12 basic solution, monomer backbone concentration range of 100 mM to 5 M, weakly acidic solution of formic acid aqueous solution with a concentration range of 50 mM to 2.5 M, incubation in a glass container at 27 degrees Celsius. Thus, the size of the self-assembled organic nanosensors ranges from 40 nm to 2000 nm.

[0046] Figure 2 shows dynamic light scattering data of self-assembled organic nanosensors. The nanosensors were synthesized using a 500 mM monomer backbone solution in base and a 50 mM ascorbic acid solution in a plastic container at 57 degrees Celsius in the dark for 72 hours. The samples were analyzed using a Photocor Complex instrument from Photocor Instruments, Estonia. The size of the nanosensors was determined from the particle size distribution analysis obtained from the autocorrelation function, which showed a major peak between 220-360 nm with a goodness of fit c 2 = 0.017, indicating that the size of the self-assembled organic nanosensors was between 220-360 nm, as shown in Figure 2-A. The self-assembled organic nanosensors were dispersed and the autocorrelation function decayed exponentially, indicating that the particle size distribution was between 220-360 nm (Figure 2-B), however, in the autocorrelation function of Figure 2-C, the monomer backbone did not show exponential decay due to its very small size, which indicates that the size of the monomer backbone was less than 2 nm. The monomer backbone also did not show self-assembly or aggregation in the presence of albumin, as the autocorrelation function remained similar with and without albumin, without exponential decay (Figure 2-D). Thus, the self-assembly of the organic nanosensors was facilitated by the processing of the backbone with a specific process and in the absence of albumin. Moreover, the physical size and properties of the self-assembled organic nanosensors remained stable after interaction with albumin. Figure 2E is the autocorrelation function of the self-assembled organic nanosensors with albumin, which shows similar exponential decay as the autocorrelation function of the nanosensors alone. Thus, the DLS data of the nanosensors with and without albumin remained similar, which indicates that the nanosensors were stable and did not disassemble even in the presence of albumin. The binding mode of albumin to the self-assembled organic nanosensors should be many-to-one, as opposed to the one-to-many binding mode of albumin to the monomer backbone in Figure 3.

[0047] The following protocol was used to perform absorption spectrophotometric analysis of the self-assembled organic nanosensor and the monomer backbone. The self-assembled organic nanosensor was loaded into a test tube containing 100 mM, pH 7 phosphate buffer at a weight to volume ratio of 1 : 100,000. The solution was transferred to a cuvette compatible with absorption spectrophotometry in the UV-VIS range. Absorption spectral analysis was performed in a Cary 100 from Agilent Technologies using the Cary 100 software. The absorption spectrum was determined between 250 nm and 700 nm. In both cases, two different peaks were observed for the self-assembled organic nanosensor and the monomer backbone, peak 1 between 260-280 nm with a maximum absorption at 262 nm and peak 2 between 350-380 nm with a maximum absorption at 352 nm (Figure 4). Thus, despite the difference in size between the monomer backbone and the self-assembled organic nanosensor, the electronic structure is still similar. The monomer absorbs at least 1.2 times more light than the self-assembled organic nanosensor (Figure 4).

[0048] Fluorescence spectrophotometric analysis of the self-assembled organic nanosensor is described herein. The self-assembled organic nanosensor was loaded into a test tube containing 100 mM, pH 7 phosphate buffer at a weight to volume ratio of 1 : 100,000 to 1 : 1000. The solution was transferred to a fluorescence compatible cuvette and two-dimensional fluorescence was analyzed in a Fluoromax-4 from Horiba Ltd. using the Fluoromax-4 software with an excitation wavelength range of 280 nm to 420 nm and an emission wavelength range of 400 nm to 650 nm. The fluorescence spectrum of the monomer backbone was also analyzed using the same protocol. The self-assembled organic nanosensor has a single broad excitation peak with a maximum between 350-380 nm while the monomer backbone has two excitation peaks with a maximum between 290-310 nm and 350-370 nm (Figure 5-A). The emission spectrum of the nanosensor has a single low intensity broad peak with a maximum between 525-545 nm while the monomer backbone has a single high intensity peak between 420-480 nm (Figure 5-B). Furthermore, the monomer backbone with the same weight as the nanosensor has 16 times more emission at a wavelength of 450 nm. The autofluorescence of the monomer dye increases the detection noise resulting in a low signal to noise ratio. In this way, the self-assembled organic nanosensor with low autofluorescence will have a high signal to noise ratio. Thus, the fluorescence behavior of the self-assembled organic nanosensor is different from the monomer backbone with clear differences in the peak position and peak height in the excitation and emission spectra (Figure 5).

[0049] The interaction of the self-assembled organic nanosensor and the monomeric backbone with albumin was investigated using the following method. The self-assembled organic nanosensor was loaded into a test tube containing 100 mM phosphate buffer at a ratio of 1 : 100,000 by weight by volume. To this solution, a stock solution of albumin was added at a concentration of 1 gram per liter (g / L) to a final concentration of 88 milligrams per liter (mg / L). Fluorescence analysis of the emission was performed using a fluorescence spectrophotometer Fluoromax-4 (Horiba Limited) over a wavelength range of 400 nm to 650 nm. Using the same protocol, the fluorescence spectrum of the monomeric backbone with albumin was also analyzed, where the monomeric backbone was in 100 mM phosphate buffer (pH 7) at a ratio of 1 : 10,000, and the albumin concentration used for analysis was 88 mg / L. The analysis showed that the fluorescence spectrum of the self-assembled organic nanosensor exhibited a blue shift in the presence of albumin, while the monomeric form exhibited a red shift in the presence of albumin. A shift in the spectrum from a higher wavelength to a lower wavelength upon interaction with an analyte is called a blue shift or short shift, and conversely, a shift in the spectrum from a lower wavelength to a higher wavelength upon interaction with an analyte is called a red shift or long shift. This indicates that there is a fundamental difference in the sensing principle between the monomeric backbone and the disclosed self-assembled material. The maximum emission wavelength of the autofluorescence of the self-assembled organic nanosensor was between 525 nm and 545 nm, while the maximum emission wavelength of the albumin was shifted to a lower wavelength, i.e., 460 nm to 520 nm, whereas the maximum emission wavelength of the autofluorescence of the monomeric form was 450 nm, and the maximum emission wavelength of the albumin was shifted to a higher wavelength, i.e., 475 nm (Figure 6). The relative fluorescence increase factor of the monomeric backbone with and without albumin was 3 at a wavelength of 450 nm, while the relative fluorescence increase factor of the nanosensor with and without albumin was 34.6 at a wavelength of 470 nm. Therefore, the low autofluorescence of the self-assembled organic nanosensor is beneficial for improving the signal-to-noise ratio, thereby allowing detection of lower concentrations of analytes with higher resolution.

[0050] The detection range of the self-assembled organic nanosensor has been determined to be 0.1 mg / L to 3500 mg / L of albumin. The fluorescence spectrophotometer used was a Fluorolog-3 (Horiba Limited) from Prantae Solutions Private Limited. Analysis was performed with light source wavelength of 320-400 nm and light sensor to capture emitted light. The self-assembled organic nanosensor was loaded into a test tube containing 100 mM phosphate buffer at a weight to volume ratio of 1 : 1000. Relative fluorescence units were measured. Albumin stock solution was added to the self-assembled organic nanosensor solution to achieve the desired albumin concentration. The ratio of albumin sample to self-assembled organic nanosensor solution was 1 : 10. The albumin concentrations studied to determine the range were 0.1, 1, 10, 50, 100, 500, 1000, 1500, 3000, and 3500 mg / L. The relative fluorescence values were determined using a fluorescence spectrophotometer system with light source wavelength of 320-400 nm, where the self-assembled organic nanosensor showed interaction with albumin at concentrations ranging from 0.1 mg / L to 3500 mg / L and produced detectable fluorescence, as shown in Figure 7. The fluorescence intensity was directly proportional to the concentration of albumin in aqueous solution. Beyond 500 mg / L, the same weight of monomeric scaffold became saturated. This indicates that the self-assembled organic nanosensor, with its high surface to volume ratio of the nanoparticle, is able to interact with more albumin molecules and hence a wider range of albumin detection can be observed. In addition, the same amount of monomeric scaffold was not able to distinguish concentrations below 10 mg / L. This is due to the high autofluorescence of the monomeric dye resulting in low signal to noise ratio and hence reduced resolution at low concentration regions.

[0051] The self-assembled organic nanosensor has higher specificity for albumin compared to non-albumin proteins. Figure 8 shows three different forms of albumin, namely human serum albumin, bovine serum albumin, and ovalbumin, as well as non-albumin proteins tyrosine and lysozyme, each at a concentration of 100 mg / L. The fluorescence spectrophotometer system from Prantae Solutions Private Limited was used to measure the relative fluorescence units of each sample. The self-assembled organic nanosensor showed higher fluorescence with albumin compared to non-albumin proteins. Analysis was performed with light source wavelength of 320-400 nm and light sensor to capture emitted light. Relative fluorescence units were measured, with lower or negligible fluorescence for non-albumin proteins and higher fluorescence for albumin.

[0052] The reaction time of the self-assembled organic nanosensor with albumin was less than 10 seconds. Time course study was performed by visualizing fluorescence with excitation light source at 380 nm. Bovine serum albumin at a concentration of 100 mg / L was added to the self-assembled organic nanosensor solution. Green fluorescence was visualized at 90 degrees to the incident light at 0, 5, 10, 15, 20, 30, 60, and 120 seconds. Fluorescence started to appear between 5 and 10 seconds, as shown in Table 1. Thus, the interaction between albumin and the self-assembled organic nanosensor was fast and stable over a long period of time, enabling rapid detection between 5 and 10 seconds and remaining stable up to 120 seconds during the study period.

[0053] Table 1: Time period study of fluorescence reaction between albumin and self-assembled organic nanosensor

[0054] Time period after addition Observation of fluorescence Fluorescence intensity 0 No - 5 Yes + 10 Yes ++ 15 Yes +++ 20 Yes +++ 30 Yes +++ 60 Yes +++ 120 Yes +++

[0055] Albumin complexed with self-assembled organic nanosensor is more stable than albumin in aqueous solution at 27 degrees Celsius. Reaction mixtures were prepared using self-assembled organic nanosensor in 100 mM phosphate buffer at pH 7 with a volume of 3.4 ml, where the ratio of nanosensor to buffer solution was 1 part by weight to 10,000 parts by volume. Two different concentrations of albumin were tested: 100 mg / L and 330 mg / L. Albumin at each concentration was incubated in phosphate buffer at pH 7 with and without the addition of self-assembled organic nanosensor, and the stability of fluorescence was measured daily for 20 consecutive days. The stability of albumin samples with the addition of self-assembled organic nanosensor was determined in terms of relative fluorescence values and compared to the zero day fluorescence as a reference. The stability of albumin samples incubated in buffer only was determined in terms of relative fluorescence values by the addition of nanosensor (ratio of nanosensor to buffer solution was 1 part by weight to 10,000 parts by volume) and compared to the zero day fluorescence as a reference. Relative fluorescence units were determined using a spectrofluorometer (excitation wavelength of 320-400 nm) and the emission light was captured using a light sensor at a temperature of 27 degrees Celsius for 20 days. Albumin in buffer lost 50% of its fluorescence in 1 day and more than 90% in 3 days, whereas albumin incubated with self-assembled organic nanosensor in buffer exhibited high stability with only a 4% decrease observed over the 20 days of the study (Figure 9). Thus, the nanosensor can stabilize albumin that is prone to degradation in aqueous solution at 27 degrees Celsius.

[0056] The thermal stability of self-assembled organic nanosensor was tested by incubating the nanosensor at both -80 degrees Celsius and 95 degrees Celsius for 120 minutes. The self-assembled organic nanosensor was then brought to 27 degrees Celsius and tested for fluorescence activity at 100 mg / L, 200 mg / L, and 300 mg / L under excitation at 350-400 nm wavelength. Table 2: Self-assembled organic nanosensor retained 100% activity at both negative (-) 80 degrees Celsius and positive 95 degrees Celsius.

[0057] Table 2: Thermal stability of nanosensor

[0058] Treatment temperature Fluorescence activity retained by nanosensor 27℃ 100％ -80℃ 100％ 90℃ 100％

[0059] Self-assembled organic nanosensor can be used as a kit for the measurement of albumin in environmental, biological, or synthetic samples in solid state or in solution in the field, in situ, or on the spot for testing and diagnosis. ​

[0060] The kit comprises self-assembled organic nanosensors in aqueous solution, filled in an optically transparent chamber, which can be but is not limited to a microfluidic chamber, a tube, a vial, a cuvette made of plastic, glass or quartz. The chamber can be sealed, covered or open.

[0061] Albumin sample should be added to the chamber, the volume of albumin sample can be from 1:50 to 10:1 of the self-assembled organic nanosensor solution. Albumin sample can be but is not limited to synthetic material, biological sample or environmental sample. Albumin sample should be mixed with self-assembled organic nanosensor solution by manual or automatic shaking, pipetting or vortexing. The fluorescence output of the interaction of self-assembled organic nanosensor with albumin can be observed visually or using a fluorescence spectrophotometer or imaging system, the input light should be in the wavelength range of 320-400 nm, and perpendicular to the observation angle.

[0062] In another embodiment, the kit comprises self-assembled organic nanosensors embedded in a solid matrix, wherein the solid matrix can be but is not limited to nitrocellulose membrane, filter paper, glass slide and other solid surface. The self-assembled organic nanosensors embedded in a solid matrix can be immersed in a sample containing albumin solution, or albumin solution can be poured on the self-assembled organic nanosensors embedded in a solid matrix, or protein can be spotted on the self-assembled organic nanosensors embedded in a solid matrix. The fluorescence output of the interaction of self-assembled organic nanosensors with albumin can be observed visually or using a fluorescence imaging system, the input light should be in the wavelength range of 320-400 nm, and perpendicular to the observation angle.

[0063] The following are non-limiting examples describing the efficacy of the system.

[0064] Example

[0065] Albumin standard curve preparation for guidance and training.

[0066] In 1x phosphate buffered saline solution {137 mM NaCI (Sigma Aldrich, Cat. No. S7653-250G, Batch No. SLBV9983), 10 mM KH2PO4(Merck Cat. No. 1.93605.5021, Batch No. DH0D701207), 1.8 mM Na2HPO4(Merck Standard solutions of 10 mg / mL of bovine serum albumin (from Himedia) stock were prepared in PBS. Working solutions of bovine serum albumin (BSA) were prepared in PBS at different concentrations in a volume of 1 mL. The range was determined using standard BSA analyte solutions at different concentrations (i.e. 0, 1, 2, 4, 8, 12, 16, 20, 25, 30, 45, 60 and 90 mg / dL). The relative fluorescence value was measured for each concentration using a fluorospectrophotometer (Shimadzu UV-1800) at an excitation wavelength of 380 nm. The emission light was captured using a photodetector. The plot of concentration versus relative fluorescence units was plotted to generate a standard curve. This standard curve can be used to determine the unknown concentration of albumin in unknown samples.

[0067] Evaluation of albumin in bovine urine

[0068] Urine was collected from a healthy bovine in a cattle shed and was filled in a vial. 170 microliters of urine was added to the kit and the self-assembled organic nanosensor solution was 1530 microliters in volume and was placed in an optically transparent chamber made of acrylic. The sample was mixed by shaking and was observed using a fluorospectrophotometer at an excitation wavelength of 380 nm. The relative fluorescence obtained was 2802. The fluorescence intensity was converted to concentration using the standard curve described in the previous example (Figure 10-A). The concentration of the sample (unknown) was determined to be 34 mg / L.

[0069] Evaluation of albumin in bovine milk samples

[0070] Bovine milk was collected in a container and was diluted 20 times with 1 x Phosphate Buffered Saline solution {137 mM NaCI (Sigma Aldrich, Cat. No. S7653-250G, Batch No. SLBV9983), 10 mM KH2PO4(Merck Cat. No. 1.93605.5021, Batch No. DH0D701207), 1.8 mM Na2HPO4(Merck Cat. No. 1.06585.1000, Batch No. K51666285026) at pH 7.4. The nitrocellulose membrane (by Bio-Rad Laboratories India Private Limited) impregnated with the self-assembled organic nanosensor using a slot blot apparatus was dipped in the 20 times diluted bovine milk sample for 60 seconds and was subsequently washed three times with 1 x Phosphate Buffered Saline solution. The nitrocellulose membrane was finally observed on a UV transilluminator to qualitatively detect albumin in the sample (Figure 10-B).

[0071] Evaluation of albumin in human urine samples

[0072] ​A human urine sample was collected in a container. 170 microliters of the human urine sample was added to the kit and the self-assembled organic nanosensor solution volume was 1530 microliters, which was placed in an optically transparent chamber made of acrylic. After the sample was mixed by shaking, it was observed using a fluorescence spectrophotometer at an excitation wavelength of 380 nm. The relative fluorescence obtained was 8769. The fluorescence intensity was converted to concentration using the standard curve described in the previous example (Figure 10-A). The concentration of the sample (unknown) was determined to be 500 mg / L.

[0073] Evaluation of albumin in a human serum sample

[0074] A human serum sample was collected and diluted 40 times with 1 x phosphate buffered saline solution {137 mM NaCI (Sigma Aldrich, Cat. S7653-250G, Lot SLBV9983), 10 mM KH2PO4 (Merck Cat. 1.93605.5021, Lot DH0D701207), 1.8 mM Na2HPO4 (Merck Cat. 1.06585.1000, Lot K51666285026)} at pH 7.4. 170 microliters of the human urine sample was added to the kit and the self-assembled organic nanosensor solution volume was 1530 microliters, which was placed in an optically transparent chamber made of acrylic. After the sample was mixed by shaking, it was observed using a fluorescence spectrophotometer at an excitation wavelength of 380 nm. The relative fluorescence obtained was 9546. The fluorescence intensity was converted to concentration using the standard curve described in the previous example (Figure 10-A). Taking into account the dilution factor, the concentration of the sample (unknown) was determined to be 2800 mg / L.

[0075] Beneficial effects

[0076] The beneficial effects of the self-assembled organic nanosensor are as follows.

[0077] 1. The method is low cost because the self-assembled organic nanosensor is synthesized from a low-cost naphthalenesulfonate monomer backbone.

[0078] 2. The synthesis is based on an economically efficient self-assembly process.

[0079] 3. The assembly of nanoparticles provides a high surface-to-volume ratio, enabling the detection of more analyte.

[0080] 4. The self-assembled organic nanosensor has very low autofluorescence, so the signal-to-noise ratio is better.

[0081] 5. The self-assembled organic nanosensor can detect a wide range of albumin concentrations, from 0.1 mg / L to 3500 mg / L.

[0082] 6. The self-assembled organic nanosensor is highly specific for albumin.

[0083] 7. The albumin detection using the self-assembled organic nanosensor is a simple one-step method.

[0084] 8. The fluorescence produced by the interaction of albumin with the self-assembled organic nanosensor can be observed visually, or by a fluorescence spectrophotometer or imaging system.

[0085] 9. The interaction of albumin with the self-assembled organic nanosensor is fast and remains stable for long periods of time, thus allowing for rapid detection and no loss of detection results over time.

[0086] 10. The self-assembled organic nanosensor is very stable even under extreme temperature conditions of -80°C and 95°C.

[0087] It is understood that various modifications, changes, and adaptations can be made to the present application by those skilled in the art without departing from the spirit of the present application or exceeding the scope of the claims described for illustrative purposes.

[0088] Any addition or deletion to the examples is hereby considered a modification to the specification, thus satisfying the written description requirement for all elements used in the claims.

Claims

1. A synthetic method for self-assembled organic nanosensor for albumin detection, comprising: dissolving naphthalenesulfonate monomer backbone in a basic solution having a pH in the range of 8-12 at a concentration of 250 millimolar (mM) to 4 molar (M) to obtain a monomer backbone solution having a final concentration in the range of 100 millimolar (mM) to 5 molar (M); mixing the basic solution of naphthalenesulfonate monomer backbone with an aqueous buffer solution having a pH in the range of 3-5, wherein the molar concentration of the buffer solution varies in the range of 50 millimolar (mM) to 2.5 molar (M); incubating in a closed glass or plastic container in a dark environment at a temperature in the range of 27 to 57 degrees Celsius for a period of 12 to 86 hours; centrifuging the self-assembled organic nanosensor formed as a precipitate using 10,000 rpm for a period of 10 minutes; resuspending the self-assembled organic nanosensor precipitate in deionized water.

2. The method of claim 1, wherein the self-assembled organic nanosensor is synthesized in a size range of 40 nanometers to 2000 nanometers, and an organic solvent is used along with the naphthalenesulfonate monomer backbone for synthesis of the self-assembled organic nanosensor.

3. The method of claim 1, wherein the self-assembled organic nanosensor remains stable in an aqueous solution in the temperature range of negative 80 degrees Celsius to 95 degrees Celsius.

4. The method of claim 1, wherein the self-assembled organic nanosensor is stable in the presence of the albumin and does not decompose, wherein the albumin acts on the self-assembled organic nanosensor in a multibinding mode with respect to the albumin to monomer backbone.

5. The method of claim 1, wherein the self-assembled organic nanosensor stabilizes the albumin in an aqueous solution for a period of 20 days at 27 degrees Celsius.

6. The method of claim 1, wherein the self-assembled organic nanosensor exhibits low spontaneous fluorescence emission in an aqueous solution with a peak maximum between 525-545 nm wavelength, and shows high fluorescence in the presence of albumin with a peak maximum between 460-520 nm wavelength.

7. A self-assembled organic nanosensor for albumin detection having the following molecular structure: which has a high signal to noise ratio.

8. The nanosensor of claim 7, wherein the self-assembled organic nanosensor binds albumin in a concentration range of 0.1 mg / L - 3500 mg / L and exhibits fluorescence with a peak maximum varying between 460-520 nm, and wherein the fluorescence intensity varies in proportion to the concentration of albumin.

9. The nanosensor of claim 7, wherein the self-assembled organic nanosensor interacts with the albumin in a time range of 5 to 10 seconds.

10. The nanosensor of claim 7, wherein the self-assembled organic nanosensor is embedded and bound to a solid matrix and immersed in a sample containing the albumin solution embedded in the solid matrix, wherein the interaction of the self-assembled organic nanosensor and the albumin is observed as fluorescence, and wherein the input light is in the range of 350-400 nm and perpendicular to the viewing angle.

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