CN110579461A

CN110579461A - Preparation method and application of SERS performance detection biosensor

Info

Publication number: CN110579461A
Application number: CN201910402171.XA
Authority: CN
Inventors: 丁显廷; 沈广霞; 梅穆纳·阿兹玛特
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-05-15
Filing date: 2019-05-15
Publication date: 2019-12-17

Abstract

The invention relates to a preparation method and application of a SERS performance detection biosensor. The invention discloses a simple and rapid preparation technology of a Raman enhanced active material, and a biosensor is constructed to realize detection of pesticide residues and toxic substances in liquid such as milk. The method comprises the following steps: the preparation method comprises the steps of preparation of an SERS active chip, preparation of gold nano materials with different scale diameters and different appearances, and preparation of a detection chip with optimal SERS performance formed by coupling the gold nano materials and a substrate material. The detection of melamine in liquid milk is confirmed. The method is simple, economical, rapid and sensitive, has obvious effect and high selectivity, and is suitable for large-scale popularization and production.

Description

Preparation method and application of SERS performance detection biosensor

Technical Field

The invention relates to the fields of life science and food safety, in particular to a novel simple and rapid preparation technology of a Raman enhanced active material, and a biological detection chip is constructed. The detection of pesticide residue and toxic substances in liquid such as milk and the like is realized through the biosensor coupled by the gold nano material and the substrate material.

Background

Surface-Enhanced Raman Scattering (SERS) analysis is commonly used for tracking and detecting toxic compounds, pesticides, and clinical diagnosis. While metal nanostructured SERS-active substrates generally exhibit large SERS enhancements, longer active lifetimes, better reproducibility, and fewer detection limitations. The colloidal metal polymer with good dispersibility and stability is formed in a solution as a SERS active medium. The fundamental limitation of these agglomerates is their inherent instability and poor SERS signal reproduction. Moreover, colloidal metals tend to precipitate in solution. Although some emulsifiers and deliquescent polymers promote the stability of metal particles, these agents contribute to the background signal noise that accompanies the target molecule in SERS. Therefore, it is necessary to embed colloidal metals on solid polymer matrices to minimize coagulation. In the prior art, porous nanoparticles, compared to typical non-porous substrates, can achieve greater signal levels, indicating that monoliths are more effective and sensitive for detecting traces of biomarkers or toxic additives using the monoliths as the metal nanostructure matrix.

Melamine (C)₃N₃(NH₂)₃) Melamine and protamine are triazine nitrogen-containing heterocyclic organic compounds, are used as chemical raw materials, and cannot be used for food processing or food additives. And 35, 10 and 27 in 2017, and the list of carcinogens published by the international cancer research institution of the world health organization is preliminarily collated for reference, wherein melamine is in the list of 2B carcinogens. In modern processes melamine resins are often used in food packaging or tableware, and thus the melamine in food is often considered a result of migration. In the food safety standard established by the united nations, the melamine content in each kilogram of liquid milk is regulated to be not more than 0.15 mg. However, in society, melamine is added to foods such as milk, milk powder, frozen yogurt, pet food, cookies, coffee beverages and the like by illegal merchants to increase the protein content, which causes serious food safety problems. Ten years ago, the Chinese poisonous milk powder event caused a wide concern all over the world, and a large number of pets suffered diseases or died due to eating melamine-containing feed in the United states before that. In the modern technology, the method has the advantages that,In the scientific and developing era, how to simply and effectively perform trace analysis on melamine still remains a key challenge in the field of basic life science research and food analysis.

Various efforts have been made worldwide to detect melamine in food products. Including liquid chromatography, gas chromatography, capillary electrophoresis, spectroscopy, nuclear magnetic resonance, etc., the demand for melamine detection is increasing with the variety of detection means, and a faster, more convenient and more economical method needs to be found.

In order to improve the detection capability of melamine, other pesticides and toxic liquids, the technical personnel in the field are dedicated to develop a new porous SERS active substrate which has higher sensitivity, can more conveniently and accurately detect toxic compounds in the food field, and provides a new idea for sensing biomedicine and biological analysis evaluation.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is that the existing SERS substrate has low sensitivity, low reproducibility and instability. .

In order to solve the above problems, the present invention provides a method for preparing a SERS performance detection biosensor, comprising the steps of:

step 1, preparing a SERS active chip, wherein the SERS active chip is a porous silicon wafer with a gold coating;

Step 2, preparing a gold nanoparticle solution, wherein the diameter of the gold nanoparticle is 10-100 nM;

and 3, adding the gold nanoparticle solution obtained in the step 2 to the surface of the SERS active chip obtained in the step 1, air-drying at room temperature, and thoroughly cleaning to obtain the SERS performance detection biosensor. Preferably, the thorough washing is performed using deionized water, and the addition amount of the gold nanoparticle solution is 10 μ L.

Further, the gold nanoparticles in step 2 include, but are not limited to, gold nanospheres, gold nanorods and gold nanoprisms.

Further, the gold nanosphere is prepared by the following method,

Step A, heating the sodium citrate aqueous solution to 100 ℃ and stirring vigorously while using a condenser to prevent the solvent from evaporating

Step B, mixing HAuCl₄The solution was poured dropwise and a color change was observed within 10 minutes;

Step C, when the synthesized gold seeds are cooled to 90 ℃, sodium citrate and HAuCl are added₄Sequentially injecting into the mixed solution;

And D, after 30 minutes, repeating the step C for more than 2 times to grow the gold particles to obtain the gold nanospheres, wherein the diameter of the gold nanospheres is 30-70 nM. The preferred number of times is 3, with 3 generations of growth.

Further, the aqueous solution of sodium citrate in step A was 150mL, 2.2mM, and the stirring time was 15 minutes. In the step B, the tetrachloroaururic acid solution is 1mL and 25Mm, and the color is changed from yellow to blue-gray to light powder. In step C, the concentration of sodium citrate and tetrachloroauric acid is 1mL, 60mM and 1mL, 25mM respectively. Further, the gold particles in step D were grown to about 45 nm.

Further, the preparation of the gold nanorod solution is to synthesize the gold nanorod solution by using hexadecyl trimethyl ammonium bromide as a shape-oriented surfactant, wherein the diameter of the gold nanorod is between 50 and 75 nM. Specifically comprises

synthesizing gold nanorod solution by using Cetyl Trimethyl Ammonium Bromide (CTAB) as a shape-directing surfactant, and centrifuging by using a centrifuge to disperse sediments in pure water, wherein the centrifugal speed is 9600rpm, and the time is 14 min; repeating for at least 3 times, and storing the clean gold nanorods in a refrigerator.

Further, the gold nanoprism solution is prepared by a method comprising the steps of dropwise adding a tetrachloroauric acid solution into a newly prepared sodium thiosulfate solution and a potassium iodide aqueous solution, uniformly stirring, dropwise adding the sodium thiosulfate solution into a reaction system, stirring the final solution, and removing unreacted reagents to obtain the gold nanoprism, wherein the diameter of the gold nanoprism is 30-90 nM. Preferably, the method comprises adding dropwise tetrachloroauric acid solution to the newly prepared thio-sulfideSodium acid solution (Na)₂S₂O₃) Stirring with potassium iodide (KI) aqueous solution at moderate and medium speed, wherein the concentration of tetrachloroauric acid is 10mL and 2mM, the concentration of sodium thiosulfate is 12mL and 0.5mM, and the concentration of potassium iodide aqueous solution is 32.7 mu L and 0.1M, and stirring for 9 minutes; dropwise adding a sodium thiosulfate solution into the reaction system, stirring the final solution, wherein the sodium thiosulfate is 2mL and 0.5mM, and stirring for 90 minutes; the unreacted reagents were removed to obtain gold nanoprisms, which were centrifuged and washed with pure water at 3500rpm for 3 times.

Further, in the gold nanoparticle solution, the concentration of gold nanosphere particles is 1 × 10¹³the concentration of the gold nanorods and the gold nanoprisms is 2 multiplied by 10¹⁰one/mL.

Further, the preparation of the SERS active monomer specifically comprises the following steps:

Step 1.1, preparing a silicon wafer substrate with a gold coating, cleaning and drying the silicon wafer substrate, and oxidizing the silicon wafer substrate in a low-pressure plasma cleaner; the gold-coated silicon wafer substrate is preferably cleaned with acetone, methanol and water in an ultrasonic bath, dried under nitrogen and the substrate surface further oxidized under a low pressure plasma cleaner.

step 1.2, preparing and uniformly mixing a reaction solution, wherein the reaction solution is set to enable the silicon-coated gold thin film substrate to generate a porous structure; the reaction solution was treated with ultrasound to obtain a homogeneous solution, and further treated with nitrogen to remove oxygen.

And step 1.3, adding a reaction solution to the silicon-coated gold thin film substrate, exposing the substrate under an ultraviolet light source, cleaning the surface and drying the surface. Preferably acetone, continued thorough rinsing with deionized water and drying under nitrogen;

Further, the substrate size in step 1.1 is 5X 5mm²The washing was performed 3 times in sequence, and the washing time was 5 minutes each time.

Further, the reaction solution in step 1.2 includes 24 parts by mass of glycidyl methacrylate, 18 parts by mass of ethyl dimethacrylate, 50 parts by mass of cyclohexanol, 10 parts by mass of methanol, and 1 part by mass of 2, 2-dimethylolpropionic acid.

further, the purge time in step 1.2 under nitrogen was 15 minutes.

Further, in step 1.3, the volume of the solution is 2 μ L, the incident power of the ultraviolet light source is 22.0mW/cm2, and the duration is 900s, so that the whole layer is uniformly solidified on the gold surface.

Further, the time of the step 1.3 is 30 minutes, and the surface is modified by active oxygen radicals, so that the number of the functional pole regions on the surface of the substrate is remarkably increased.

The invention also provides application of the SERS performance detection biosensor produced by the preparation method of the SERS performance detection biosensor in detection of toxic compounds, pesticides and clinical diagnosis. The use further includes applications in the fields of sensing biomedicine, bioanalytical evaluation and food safety

Further, the invention provides an application of the SERS performance detection biosensor in rapid detection of melamine content in milk.

The enhanced efficiency of the biosensor is detected by utilizing the SERS performance, the sensitivity and the selectivity are higher, a long extraction or analysis program is not needed, the whole detection and analysis process can be completed within 10 minutes, and a new idea is provided for detecting melamine in milk, so that the sensing biomedicine and the biological analysis evaluation are provided.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a transmission electron micrograph of three gold nanoparticles;

FIG. 2 is a UV-VIS absorption spectrum and typical peaks of three gold nanoparticles;

FIG. 3 is a FTIR broad spectrum of three gold nanoparticles;

FIG. 4 is an SEM image of a SERS performance measuring biosensor;

FIG. 5 is a schematic representation of the detection of a SERS performance detecting biosensor;

Fig. 6 is a comparison of raman spectra of different gold nanoparticles under the same conditions for R6G;

Figure 7 is GNS @ GEMS for the determination of melamine capacity and range in liquid milk.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Detailed Description

The invention is further described below with reference to the drawings and the embodiments.

example 1

1. Preparation of SERS active chip

Prior to chip preparation, gold-coated 5 × 5mm 2-sized silicon wafer substrates were cleaned in the given order with acetone, methanol and water in an ultrasonic bath for 5 minutes and dried under nitrogen. Further oxidation is carried out under low pressure plasma cleaner to increase the surface energy and make the surface hydrophilic.

Preparing a reaction solution including 24 parts by mass of glycidyl methacrylate, 18 parts by mass of ethyl dimethacrylate, 50 parts by mass of cyclohexanol, 10 parts by mass of methanol and 1 part by mass of 2, 2-dimethylolpropionic acid, obtaining a uniform solution by ultrasonic treatment, and further purging the solution with nitrogen to remove oxygen; placing 2 μ L of the reaction solution on the surface of the cleaned and dried substrate; at an incident power of 22.0mW/cm²Exposing for 900s to solidify the whole layer on the surface of gold; thoroughly rinsing the surface of the monolith with acetone, then thoroughly rinsing with deionized water and drying under nitrogen; finally, the substrate is placed into the plasma cleaning machine again for 30 minutes, and the surface of the polymer monolith is modified by active oxygen radicals, so that the number of the functional pole areas on the surface of the substrate is obviously increased.

Example 2

Preparation of gold nano-materials with different scale diameters and different morphologies

150mL of a 2.2mM aqueous solution of sodium citrate was heated to 100 ℃ and held for 15 minutes with vigorous stirring. At the same time, use coldA condenser to prevent evaporation of the solvent. Then, 1mL, 25mM tetrachloroauric acid (HAuCl)₄) The solution was poured dropwise. The color of the solution gradually changed from yellow to blue-gray and then to light pink within 10 minutes. When the synthesized gold seed crystal was cooled to 90 ℃, 1mL of sodium citrate (60mM) and another 1mL of tetrachloroauric acid (25mM) solution were sequentially injected into the mixed solution. After 30 minutes, the process was repeated (1 mL, 60mM repeated sodium citrate and 1mL, 25mM tetrachloroauric acid added sequentially) until 3 generations and gold particles were grown to about-45 nm, yielding Gold Nanoparticles (GNSs).

Gold Nanorods (GNRs) were prepared according to a seed-mediated growth method. Cetyl trimethylammonium bromide (CTAB) was used as the shape directing surfactant. The synthesized gold nanorod solution was centrifuged at 9600rpm for 15 minutes by a centrifuge to remove CTAB, and the deposit was dispersed in pure water. This procedure was repeated at least three times. Finally, the clean gold nanorods were stored in a refrigerator (-4 ℃). The resulting stock solution had an estimated concentration of about 2X 10¹⁰one/mL (particles/mL).

10mL of 2mM chloroauric acid solution was added dropwise to 12mL of freshly prepared 0.5mM sodium thiosulfate solution (Na) with moderate agitation₂S₂O₃) 32.7. mu.L of a 0.1M aqueous solution of potassium iodide was neutralized and gently stirred for 9 minutes. An additional 2mL of 0.5mM sodium thiosulfate solution was added dropwise to the reaction and the final solution was stirred for 90 minutes. Finally, the unreacted reagents were removed and the Gold Nanoprism (GNPs) product was washed with pure water by a centrifugation process of 3500 cycles three times. The resulting stock solution had an estimated concentration of about 2X 10¹⁰one/mL (particles/mL).

the GNSs concentration was optimized and selected to be 1 × 10 per ml based on raman enhancement effect¹³And (4) granules. The same particle concentration was used for GNRs and GNPs.

Example 3

Preparation method of detection chip with optimal SERS performance formed by coupling gold nano material and substrate material

a concentration of 10 μ L from all three gold nanoparticle solutions was added to SERS-active chips (including blank control) separately and air dried at room temperature (25 ℃) and then thoroughly washed with deionized water.

Example 4

Melamine sampling

The melamine is mixed into the milk according to the range of 13mg/L to 0.0125mg/L and in different proportions. 400 μ L of 1M hydrogen chloride (HCl) was added to 5 ml of the spiked milk and mixed vigorously for 10 seconds. The mixture was sonicated for 15 minutes and centrifuged at 2000rpm for 30 minutes. The supernatant was collected and filtered through a 0.22 μ M PTFE filter. The pH of the filtered solution was adjusted by adding 100. mu.L of 1M sodium hydroxide. Then 10mL of pure Acetonitrile (ACN) was added to induce precipitation of proteins and fatty compounds in the solution. The solution was centrifuged at 1000rpm to separate any further aggregates for 15 minutes. The same method was used to treat melamine-free milk to prepare a blank standard. For SERS detection, 5. mu.L of supernatant was used.

Example 5

Characterization and Properties of three shapes of gold nanoparticles

FIGS. 1A-C show Transmission Electron Microscope (TEM) images of three gold nanoparticles, showing the shape and size characteristics of the selected gold nanoparticles, which is helpful for further chip study. The sizes of the gold nano-microspheres, the gold nano-rods and the gold nano-prisms are respectively about 50 +/-5 nm, 60 +/-5 nm and 60 +/-20 nm.

Fig. 2 shows the uv-vis absorption spectrum and typical peaks of gold nanoparticles. Peaks centered at 530,725 and 845nm in the UV-Vis analysis show the formation of GNS, GNR and GNPr according to their respective sizes.

FIG. 3 is an FTIR spectrum showing gold nanoparticles at 3402,2918,1649 and 1395cm^-1Different absorption bands of (c).

Fig. 4A-D are SEM images, all taken at magnifications of × 50,000 (gold nanoparticles) and × 10,000 (bulk). The results reveal the surface morphology of the nanoparticles on the porous substrate. We found that GNSs aggregates are trapped within the porous structure, while the structure of GNRs and GNPrs in the integral framework is removed.

Fig. 5 is an experimental schematic diagram of the detection principle of the SERS performance detection biosensor of the present invention, which takes gold nanospheres as an example for detecting melamine in milk, and has strong sensitivity.

Deposit 2. mu.L of rhodamine (R6G) solution (10)^-5m) was placed on the chip with a coverage area of about 2mm square and dried for 2 minutes at room temperature. Three gold nanoparticles (microspheres, rods and prisms) with the plasma peak maximum values of 530nm, 725nm and 845nm are coupled with a substrate material, and the synthesized sensor is used for respectively carrying out SERS analysis on the R6G spectrum. Among them, GNS @ GEMS (biosensor formed by coupling gold nanospheres with a substrate material) is selected as a potential SERS enhanced sensor, which can provide the highest sensitivity for detecting analytes. Melamine toxicity in liquid milk can be determined using GNS @ GEMS.

For comparison purposes, raman spectra of the analyte solution and the blank substrate were collected under the same conditions. The background noise of the blank substrate was too weak to interfere with the R6G peak, even at lower concentrations. All three nanoparticles showed the same R6G peak with different intensities, as in fig. 6.

example 6

Detection of Melamine Using GNS @ GEMS

The ability of GNS @ GEMS to detect melamine in liquid milk was demonstrated by adding 5 μ L of standard melamine-added liquid milk (10mg/L) drop-wise, as compared to a standard concentration of 10mg/L melamine in pure water, as shown in FIG. 7A. In order to determine the melamine concentration to different degrees, liquid milk is obtained by means of extraction measures. Extraction is a necessary process to detect low levels of melamine in milk, as protein and carbohydrate content prevent SERS analysis detection due to its complex chemical structure. Acidic extraction with HCl (1M) therefore provides the highest sensitivity. HCL is used to precipitate casein from milk. For further filtration and solvent extraction, acetonitrile ACN was used to induce precipitation of proteins and other compounds.

various concentrations of melamine were incorporated into milk and then analyzed and analyzed using sensors manufactured by SERS. As in fig. 7B, five concentrations (0,0.125,0.5,1.62,3.2,6.5mg/L) of melamine were analyzed. 710cm^-1With decreasing melamine concentration in the full-cream sampleAnd decreases. The blank sample without melamine at 0mg/L showed almost zero peaks, which was considered as a standard for further analysis. According to previous studies, a characteristic peak of ACN must appear at 922cm^-1As an internal reference, but in our example, the peak disappeared in the subsequent analytical tests due to the volatility of ACN.

FIG. 7C shows a linear regression model constructed as a correlation using 710cm^-1The intensity of the raman band of (a) determines the concentration of melamine. The model shows that the melamine concentration in whole milk has a good linear relationship between 0.125mg/L and 6.5 mg/L. Determination coefficient (R) of the regression model²) Is 0.99. LOD through 713cm^-1Three times the standard deviation of the raman intensity at (a) was calculated and found to be 0.11mg/L. A recent study showed that LOD and LOQ of melamine in milk were 0.012mmol/L and 0.039mmol/L (equivalent to 1.15mg/L and 4.91mg/L) for an analysis time of 20 minutes using a molecularly imprinted polymer as the SERS active substrate. By using GNS @ GEMS we were able to achieve the lowest LOD and LOQ (0.11mg/L. and 0.38mg/L) over a 10 minute time span. Our method is therefore suitable for melamine detection at concentrations ranging from 0.125mg/L to 6.25mg/L, meeting the U.S. and Chinese law limits of 1mg/L in milk base average recoveries were determined in milk samples at 0.125,1.62 and 6.5mg/L normalized concentrations and found to be 94.99%, 96.91% and 99.94%, respectively. The repeatability of the procedure over a day is called relative standard deviation (RSD%), and the accuracy is determined by calculating the% deviation. The RSD values for the 0.1,1.6 and 6.5mg/L spiked melamine samples were 8.7,1.3 and 0.12%, respectively. It can be observed that the sensor has good accuracy throughout the day.

the foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. a preparation method of a SERS performance detection biosensor is characterized by comprising the following steps:

step 2, preparing a gold nanoparticle solution, wherein the diameter of the gold nanoparticle is between 10 and 100 nm;

And 3, adding the gold nanoparticle solution obtained in the step 2 to the surface of the SERS active chip obtained in the step 1, air-drying at room temperature, and thoroughly cleaning to obtain the SERS performance detection biosensor.

2. The method of preparing the SERS performance detecting biosensor according to claim 1, wherein the gold nanoparticles in step 2 include, but are not limited to, gold nanospheres, gold nanorods and gold nanoprisms.

3. the method of preparing the SERS performance measuring biosensor as recited in claim 2, wherein the gold nanospheres are prepared by the following method,

Step A, heating a sodium citrate aqueous solution to 100 ℃ and violently stirring, and simultaneously using a condenser to prevent a solvent from evaporating;

Step B, mixing HAuCl₄dropwise adding into the solution;

And D, after 30 minutes, repeating the step C for more than 2 times to grow the gold particles to obtain the gold nanospheres, wherein the diameter of the gold nanospheres is 30-70 nM.

4. The method for preparing the SERS performance detecting biosensor according to claim 2, wherein the gold nanorod solution is prepared by synthesizing the gold nanorod solution with cetyl trimethyl ammonium bromide as a shape-oriented surfactant, and the diameter of the gold nanorod is between 50 nm and 75 nm.

5. the method for preparing the SERS performance measuring biosensor according to claim 2, wherein the gold nanoprism solution is prepared by dropping a tetrachloroauric acid solution into a newly prepared sodium thiosulfate solution and a potassium iodide aqueous solution, stirring them uniformly, dropping a sodium thiosulfate solution into the reaction system, stirring the final solution, and removing unreacted reagents to obtain the gold nanoprism having a diameter of 30 to 90 nm.

6. The method of preparing the SERS performance detecting biosensor according to claim 2, wherein the concentration of the gold nanosphere particles in the gold nanoparticle solution is 1 x 10¹³The concentration of the gold nanorods and the gold nanoprisms is 2 multiplied by 10¹⁰one/mL.

7. the method for preparing the SERS performance measuring biosensor according to claim 1, wherein the preparing the SERS active monomer comprises the following steps:

Step 1.1, preparing a silicon wafer substrate with a gold coating, cleaning and drying the silicon wafer substrate, and oxidizing the silicon wafer substrate in a low-pressure plasma cleaner;

step 1.2, preparing and uniformly mixing a reaction solution, wherein the reaction solution is set to enable the silicon-coated gold thin film substrate to generate a porous structure;

And step 1.3, adding a reaction solution to the silicon-coated gold thin film substrate, exposing the substrate under an ultraviolet light source, cleaning the surface and drying the surface.

8. The method for preparing the SERS performance measuring biosensor according to claim 7, wherein the washing and drying in step 1.1 are specifically: washed sequentially with acetone, methanol and water, respectively, in an ultrasonic bath and dried under nitrogen.

9. The method for preparing the SERS performance measuring biosensor according to claim 2, wherein the reaction solution in step 1.2 comprises 24 parts by mass of glycidyl methacrylate, 18 parts by mass of ethyl dimethacrylate, 50 parts by mass of cyclohexanol, 10 parts by mass of methanol and 1 part by mass of 2, 2-dimethylolpropionic acid.

10. The SERS performance measuring biosensor manufactured by the method of manufacturing the SERS performance measuring biosensor according to any one of claims 1 to 9, for use in the detection of toxic compounds, pesticides and clinical diagnostics.