An instant color developing wet wipe for protein residue detection and methods of making and using the same

Abstract

The application provides an instant color developing wet wipe for protein residue detection and a preparation and use method thereof. The preparation method comprises the following steps: heat sealing a composite film to form a C area, and fixing a detection substrate in the C area, one corner of the substrate is separated from a hydrophobic background control area by heat pressing or a weak welding process, heat sealing is performed to form an A area and a B area, and a separation seal is arranged between the A area and the B area; a concentrated solution is filled in the A area, the upper edge of the A area is heat sealed and closed, a diluent is filled in the B area, and the bag body is completely heat sealed and closed; the concentrated solution comprises a concentrated functional solution of Masson blue G-250, the diluent is an acidic solution containing NaCl, a phosphoric acid-dihydrogen phosphate buffer system or a citric acid-citrate buffer system is used, and 0.05-0.5M NaCl is added. The application solves a series of key technical problems such as long-term storage, instant color development, resistance to alkaline interference, high sensitivity and accurate judgment.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to an instant color-developing wipe for detecting protein residues and its preparation and usage methods. Background Technology

[0002] Protein residues are common contaminants in cleaning validation processes at medical, food processing, biopharmaceutical, and laboratory settings. Residual proteins on surfaces after cleaning can cause cross-contamination, allergic reactions, or affect subsequent experimental results. Therefore, rapid, sensitive, and reliable detection of surface protein residues is of significant practical value.

[0003] Currently, classic laboratory methods for protein quantification include the Bradford method (Coomassie Brilliant Blue G-250 dye binding method), the BCA method, the Lowry method, and ultraviolet absorption methods. Among these, the Bradford method is widely used due to its simplicity, high sensitivity (detection range 1-50 μg), and low cost. The principle of this method is that under acidic conditions, Coomassie Brilliant Blue G-250 binds to basic amino acids such as arginine and lysine, as well as hydrophobic regions in proteins, shifting its maximum absorption wavelength from 465 nm to 595 nm, and changing the solution color from light brown to bright blue. In addition, some ready-to-use rapid protein detection swabs (such as Pro-Clean) are available on the market. TM (etc.), whose principle is also based on dye binding and color development. However, these products usually require puncturing the reagent chamber for mixing before use, or the operation steps are complicated, and they generally have defects such as short shelf life and insufficient detection sensitivity (usually the lower limit is 5-10 μg), which makes it difficult to meet the requirements of cleaning validation for high sensitivity and convenient long-term storage.

[0004] The main shortcomings of existing technologies are as follows: 1. Inability to perform immediate on-site testing: The traditional Bradford method requires preparing a working solution, waiting 5-10 minutes for color development, and using a spectrophotometer to read the absorbance, making it impossible for non-professionals to obtain results immediately at the cleaning site. Although commercially available rapid swabs can be used on-site, the color development time is still relatively long or requires additional instruments for interpretation.

[0005] 2. Short shelf life of working solutions: Bradford working solutions (diluted reagents) are only stable for a few weeks at room temperature, while concentrated stock solutions can be stored for a longer period of time, but they need to be diluted before use and cannot be provided as ready-to-use products.

[0006] 3. Existing wet wipes have limited functionality: Commercially available cleaning wipes only provide cleaning functions and cannot provide results for detecting protein residues. Some test swabs (such as ATP test swabs) can provide information on microbial contamination, but cannot directly detect proteins.

[0007] 4. Results reliability is affected by interfering substances and lacks a control: Different detection methods have varying tolerances to interfering substances such as cleaning agents, salts, and reducing agents. Although the Bradford method is well tolerant to most interfering substances, users lack specific guidance; furthermore, rapid on-site detection often relies on visual colorimetry, lacking an effective background control, and is prone to misjudgment due to ambient light or individual visual differences.

[0008] Therefore, developing a protein residue detection wipe that can be stored for a long time, activated immediately before use, develops color upon wiping, has high sensitivity and reliable results, and has reasonable control methods is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for instant colorimetric wipes for protein residue detection, as well as the preparation and use methods thereof.

[0010] The present invention is specifically implemented using the following technical solutions: The present invention discloses a method for preparing an instant colorimetric wipe for detecting protein residues, comprising the following steps: S.1 Partitioning: The composite film is heat-sealed to form three independent regions: region A, region B, and region C. Isolation strips are set between adjacent regions A, B, and C. The detection substrate is fixed in region C. A hydrophobic background control region is separated from one corner of the detection substrate by hot pressing or weak welding process. S.2 Filling: Fill the concentrated solution in area A and the diluted solution in area B. After filling, completely heat-seal the bag. The concentrated solution contains Masbrilliant Blue G-250 concentrated functional solution, and the diluted solution is an acidic solution containing NaCl, using a phosphate-dihydrogen phosphate buffer system or a citric acid-citrate buffer system, and adding 0.05-0.5M NaCl.

[0011] Furthermore, the phosphate-dihydrogen phosphate buffer system contains 0.2M NaH2PO4, and the pH is adjusted to 2.0-3.0 with H3PO4; the citric acid-citrate buffer system contains 0.1M citric acid solution and 0.1M sodium citrate solution, and the pH is controlled to 2.0-3.0 by adjusting the ratio of the two solutions.

[0012] Furthermore, the concentrate contains 0.1-0.5% w / v Coomassie Brilliant Blue G-250, 30-35% v / v 95% ethanol, and 55-65% v / v 88% phosphoric acid.

[0013] Furthermore, the volume ratio of the concentrate to the diluent is 1:10 to 1:25.

[0014] A second aspect of the present invention provides an instant color-developing wet wipe for protein residue detection prepared by the above method.

[0015] Furthermore, the wet wipes include a flexible packaging bag made of multi-layer composite aluminum foil or high-barrier plastic film. The packaging bag is divided into three independent sealed chambers, namely area A, area B and area C. Area A stores a concentrated liquid, area B stores a diluent, and area C contains a detection substrate, which includes a separated background control area.

[0016] Furthermore, an isolation seal is provided between adjacent areas of Zone A, Zone B and Zone C. The isolation seal is a bendable heat seal or a weak weld seal with a micro-groove structure.

[0017] A third aspect of the present invention provides a method for protein detection using the aforementioned instant color-developing wipes, characterized by comprising the following steps: K.1 Gently bend along the isolation seals of areas A and B to break the isolation seals, allowing the concentrated functional liquid in area A to mix with the diluted liquid in area B. Shake gently several times to ensure even mixing, and the liquid will turn light brown. K.2 Squeeze one side of the mixed A / B area to allow the liquid pressure to break through the isolation seal of the C area. The mixed working liquid flows into the C area through the micro-groove. Let it stand for 5-10 seconds to ensure that the test substrate is fully and evenly wetted except for the background control area. K.3 Tear open the packaging bag along the easy-tear opening, take out the soaked test substrate, pinch the discolored area of ​​the test substrate or wipe the surface to be tested directly with a non-woven cloth. K.4 Observe the color change of the test substrate within 30 seconds. Compare the color of the wet discolored area with the color of the unwetted background control area in a corner of the test substrate or with the provided standard color chart. If the wet wipe remains a light brown similar to the background control area or shows no obvious change, it indicates that the protein residue is below the detection limit. If a bright blue color is visible to the naked eye, it indicates that there is protein residue on the surface. Based on the depth of the blue, further semi-quantitative analysis can be performed.

[0018] Furthermore, the semi-quantitative method is as follows: according to the reference colorimetric card: if the color is light blue, the protein content is 1-5 μg; if the color is bright blue, the protein content is 5-50 μg; if the color is dark blue, the protein content is greater than 50 μg.

[0019] The fourth aspect of the present invention provides the application of the instant color-developing wipes or method described above in the detection of proteins.

[0020] The present invention has the following beneficial effects: (1) The present invention provides an isolated packaging design that mixes the concentrated stock solution and the diluted solution immediately before use, ensuring the long-term stability of the product during its shelf life (≥12 months).

[0021] (2) This invention enables the immediate generation of stable, visible color change after wiping under non-laboratory conditions, without the need for additional waiting or instruments. It is the first to achieve high tolerance of wet wipes to alkaline residues without reducing detection sensitivity, reaching or exceeding the detection limit (1 μg protein) of the traditional Bradford method, thus meeting the requirements for cleaning validation.

[0022] (3) This invention achieves high repeatability of test results and broad tolerance to common interfering substances in clean environments, clarifies the technical problem of "anti-alkaline interference", provides a systematic solution through formulation design, and provides an effective background control to reduce visual interpretation error.

[0023] (4) The present invention adopts a three-zone isolation packaging and integrated background comparison design, which is conducive to achieving long-term stability and accurate interpretation of the product. The integrated background comparison is introduced for the first time, eliminating the interpretation error caused by differences in ambient light and vision.

[0024] (5) The present invention forms a non-wetting background reference area on the wet wipe body through local hydrophobic treatment, eliminating the dependence on external printed color cards, providing a reference under the same substrate and the same light environment, and significantly improving the accuracy of visual interpretation.

[0025] (6) Through the synergistic innovation of “high buffer capacity anti-alkaline interference formula” and “three-chamber isolation packaging”, this invention has for the first time successfully transformed the classic Bradford protein detection principle into a ready-to-use wet wipe product that can resist the interference of alkaline residues in real cleaning scenarios. It solves a series of key technical problems such as long-term storage, instant color development, anti-alkaline interference, high sensitivity and accurate interpretation, and has significant creative, practical and industrial value. Attached Figure Description

[0026] Figure 1 This is a cross-sectional structural diagram of the wet wipe packaging bag of the present invention. The isolation area is provided by an easily breakable heat seal.

[0027] Figure 2 This is a schematic diagram illustrating the effect of using the wet wipes of the present invention (the method used for isolating areas is an easily breakable heat-sealing strip).

[0028] Figure 3 This is a cross-sectional structural diagram of the wet wipe packaging bag of the present invention. The isolation area is sealed with a weak welding strip.

[0029] Figure 4This is a schematic diagram illustrating the effect of the wet wipes of the present invention (the method used for isolating the area is weak welding seal). Detailed Implementation

[0030] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0031] Example 1: Overall Product Structure As attached Figure 1 and 3 As shown, the product of this invention includes a flexible packaging bag made of multi-layer composite aluminum foil or high-barrier plastic film, with three independent sealed chambers and a specific isolation structure formed internally by heat sealing: Zone A (Permanent Functional Solution Storage Area): This area stores Coomassie Brilliant Blue G-250 concentrated functional solution (i.e., Bradford stock solution), which can be stored stably at room temperature for a long period of time.

[0032] Zone B (Diluent Storage Zone): This zone stores a diluent for diluting the concentrated functional liquid, preferably an acidic aqueous solution containing NaCl, to facilitate the rapid formation of the desired color development environment after mixing.

[0033] Zone C (Swipe Substrate Storage Area): This area contains a test substrate (hydrophilic nonwoven fabric) for wiping. One corner of the test substrate is separated into a hydrophobic background control area by hot pressing or weak welding. This area will not be wetted by the working fluid during testing and will always maintain its dry original color, serving as an internal reference for color comparison.

[0034] like Figure 1 As shown, an isolation structure 1, namely an isolation seal, is installed between adjacent areas A, B, and C. This isolation seal is a flexible, heat-sealed strip that can be bent and broken. Figure 2 The isolation seal has a pre-made V-shaped notch line, which the user can break by simply bending the packaging, allowing the liquids in the two areas to mix.

[0035] Keep sealed under normal storage conditions; when the mixing zone is compressed, the liquid pressure can break through the isolation seal and enter the adjacent area.

[0036] like Figure 3As shown, an isolation structure 2, or isolation seal, is installed between adjacent areas of zones A, B, and C. This isolation seal is a weakly welded seal with a micro-groove structure. This micro-groove structure does not rely on the overall bending deformation of the packaging material, but rather achieves opening through directional extrusion. This structure has a certain degree of sealing, isolating liquids in adjacent chambers and preventing cross-contamination. Figure 4 When users need to mix liquids, there is no need to bend the packaging. Instead, they can apply moderate pressure to the micro-groove seal area from the outside of the packaging with their fingers. The weak weld seal will open channels, allowing the liquids to mix.

[0037] Example 2: Functional Liquid Formulation The traditional Bradford method relies heavily on a strongly acidic environment (pH < 1.5) to ensure that Coomassie Brilliant Blue G-250 binds to proteins in a cationic form. However, in cleanliness verification scenarios, alkaline cleaning agents (such as NaOH and Na2CO3) often remain on the test surface. These are extracted by wet wipes and neutralize the acidity of the system, leading to an increase in pH and causing two major problems: ① the dye deprotonates and cannot bind to proteins (false negative); ② the free dye changes color (purple-blue), resulting in abnormal background (false positive). Existing products lack effective solutions to these problems.

[0038] This invention systematically solves the above problems through a synergistic formulation of "high-concentration acid stock solution in zone A + high-buffer capacity acidic diluent in zone B".

[0039] Area A Concentrated Functional Solution (Bradford Stock Solution): Formula: Coomassie Brilliant Blue G-250 300-400mg (preferably 350mg), 95% ethanol 100mL, 88% phosphoric acid 200mL, add deionized water to make up to 300mL.

[0040] Final concentration range: Coomassie Brilliant Blue G-250 0.1-0.5% (w / v), ethanol 30-35% (v / v), phosphoric acid 55-65% (v / v).

[0041] Function: Provides a high concentration of acid (phosphoric acid) and dye to ensure that the strong acid base is maintained after mixing with Zone B.

[0042] Diluent in Zone B: Formulation: Phosphate-dihydrogen phosphate buffer system or citric acid-citrate buffer system (e.g., 0.2M NaH2PO4, adjusted to pH 2.0-3.0 with H3PO4), and add 0.05-0.5M (preferably 0.2M) NaCl, with deionized water as the solvent.

[0043] This formula has the following advantages: (1) High buffering capacity: Unlike the use of pure water, dilute acid or simple PBS in the prior art, the B region of this invention uses a high concentration of acid-salt buffer pair, which gives it an acid buffering capacity far exceeding that of conventional working solutions. When mixed with the A region, even if a certain amount of alkaline residue is mixed into the final working solution, the pH of the system can still be stabilized in the effective color development range of 1.0-1.8.

[0044] (2) Ionic strength regulation: High concentrations of NaCl can shield cations in alkaline substances (such as Na+). + K + It can prevent electrostatic interference with dye-protein binding, while promoting the exposure of hydrophobic regions of proteins, enhancing binding efficiency, and synergistically improving colorimetric sensitivity and stability.

[0045] Working solution mixing ratio: The volume ratio of the contents of zone A to zone B is 1:10 to 1:25, preferably 1:16.7 (e.g., 2 mL of zone A and 33.4 mL of zone B). After mixing, a light brown Bradford working reagent with high resistance to alkaline interference is formed.

[0046] Example 3: Brief Description of Packaging Bag Preparation Method First, the composite film is heat-sealed at an appropriate location to form zone C and fix the internal testing substrate (non-woven fabric). Simultaneously, an independent background control zone with a hydrophobic coating is formed using a hot-pressing mold. After placing the testing substrate (non-woven fabric) inside, a heat-sealed isolation seal 1 (with a pre-reserved folding notch) is formed between A and B. Next, the concentrated solution in zone A is filled, and the upper edge of zone A is heat-sealed. Finally, the diluted solution in zone B is filled, and the bag is completely heat-sealed. The resulting product is water-proof, oxygen-proof, and light-proof, ensuring long-term stability.

[0047] Example 4: Usage method (e.g.) Figure 2 , Figure 4 ) Step 1: The user holds both ends of the packaging bag and gently bends it along the easy-break seal to break the isolation seal, allowing the concentrated functional liquid in area A to mix with the diluted liquid in area B. Shake gently several times to mix evenly, and the liquid will turn light brown.

[0048] Step 2: Squeeze one side of the mixed A / B area with your fingers to allow the liquid pressure to break through the isolation seal. The mixed working liquid will flow into the C area through the micro-groove. Let it stand for 5-10 seconds to ensure that the nonwoven fabric is fully and evenly wetted (except for the background control area).

[0049] Step 3: Tear open the packaging bag along the easy-tear opening and remove the soaked test substrate. Hold the discolored area of ​​the test substrate or directly wipe the surface to be tested with the test substrate (wipe an area of ​​about 10×10 cm with even pressure).

[0050] Step 4: Immediately (within 30 seconds), observe the color change of the test substrate. Compare the color of the wetted discolored area with the color of an unwetted control area of ​​the test substrate, or with the provided standard color chart: If the test substrate remains a light brown color similar to the background control area or shows no obvious change, it indicates that the protein residue is below the detection limit (negative, <1μg). If a bright blue color is visible to the naked eye, it indicates the presence of protein residue on the surface (positive). Further semi-quantitative analysis can be performed based on the intensity of the blue: light blue corresponds to approximately 1-5 μg of protein, bright blue to 5-50 μg of protein, and deep blue to >50 μg of protein.

[0051] The entire testing process requires no waiting and no instruments, and can be completed from mixing to interpretation within 10 seconds.

[0052] Example 5: Color Development Time and Stability Test Bovine serum albumin (BSA) at concentrations of 0, 1, 5, 10, and 50 μg was added dropwise to a clean stainless steel plate and allowed to dry. After wiping with the wet wipes of this invention, color changes were recorded at 5 seconds, 15 seconds, 30 seconds, 1 minute, and 2 minutes. The results showed that for ≥1 μg of BSA, a distinguishable blue color appeared within 5 seconds of wiping, the color stabilized at 30 seconds, and there was no significant fading or deepening within 2 minutes; the 0 μg control group remained a light brown throughout. The colorimetric reaction was rapid and stable, proving the feasibility of "instant color development."

[0053] Example 6: Detection Sensitivity When BSA was tested using the above method, the blue color produced by 1 μg of protein could be clearly distinguished visually, with a color depth significantly different from the negative control. Furthermore, in a blind test by 10 independent observers, the correct detection rate was 100%. This sensitivity is comparable to the traditional Bradford method in the laboratory (lower limit of 1 μg) and superior to commercially available protein detection swabs (typically 5-10 μg).

[0054] Example 7: Resistance to alkaline interfering substances test: The following test groups were set up on clean stainless steel plates.

[0055] Negative control group: No protein, only interfering substances.

[0056] Positive control group: 1 μg BSA, no interfering substances. Experimental group: 1 μg BSA + different concentrations of alkaline interfering substances (final NaOH concentrations of 1 mM, 5 mM, and 10 mM; final Na2CO3 concentrations of 5 mM and 10 mM).

[0057] Control group: Using conventional Bradford working solution (freshly prepared according to classic literature), 1 μg BSA + 5 mM NaOH was tested.

[0058] Wipe the corresponding areas with the wet wipes of this invention (activated according to the standard method) and the traditional working solution (dropped onto a blank non-woven fabric), respectively, and observe and record the color change with the naked eye after 30 seconds.

[0059] The results are shown in Table 1 below: Table 1

[0060] This invention's wet wipes, through the design of a high-buffer capacity acidic diluent, successfully eliminate interference from alkaline residues, the most troublesome factor in real-world cleaning scenarios. They can accurately detect 1 μg of protein even in the presence of 5 mM NaOH or 5 mM Na₂CO₃, while the traditional Bradford method is completely ineffective. This is a technological breakthrough that neither existing commercially available protein swabs nor traditional methods have achieved.

[0061] Example 8: Tolerance and Repeatability of Interfering Agents Testing was conducted on a 1 μg BSA sample containing common interfering agents: 1M NaCl, 1M KCl, 10% glycerol, 10% ethanol, 10mM DTT, 1M 2-mercaptoethanol, 0.125% Triton X-100, 0.125% Tween-20, and 3M urea. The results showed no significant interference, consistent with the control without interfering agents. Intra-batch and inter-batch repeatability tests using BSA standards showed an intra-batch coefficient of variation (CV) of approximately 5.3% and an inter-batch CV of approximately 5.4%, meeting the repeatability requirements for cleaning validation.

Claims

1. A method for preparing instant colorimetric wipes for protein residue detection, characterized in that, Includes the following steps: S.1 Partitioning: The composite film is heat-sealed to form three independent regions: region A, region B, and region C. Isolation strips are set between adjacent regions A, B, and C. The detection substrate is fixed in region C. A hydrophobic background control region is separated from one corner of the detection substrate by hot pressing or weak welding process. S.2 Filling: Fill the concentrated solution in area A and the diluted solution in area B. After filling, completely heat-seal the bag. The concentrated solution contains Masbrilliant Blue G-250 concentrated functional solution, and the diluted solution is an acidic solution containing NaCl, using a phosphate-dihydrogen phosphate buffer system or a citric acid-citrate buffer system, and adding 0.05-0.5M NaCl.

2. The preparation method according to claim 1, characterized in that, The phosphate-dihydrogen phosphate buffer system contains 0.2M NaH2PO4, and the pH is adjusted to 2.0-3.0 with H3PO4; the citric acid-citrate buffer system contains 0.1M citric acid solution and 0.1M sodium citrate solution, and the pH is controlled to 2.0-3.0 by adjusting the ratio of the two solutions.

3. The preparation method according to claim 1, characterized in that, The concentrate contains 0.1-0.5% w / v Coomassie Brilliant Blue G-250, 30-35% v / v 95% ethanol, and 55-65% v / v 88% phosphoric acid.

4. The preparation method according to claim 1, characterized in that, The volume ratio of the concentrate to the diluent is 1:10 to 1:

25.

5. An instant color-developing wipe for protein residue detection prepared by any one of the methods described in claims 1-4.

6. The instant color-developing wet wipes as described in claim 5, characterized in that, The invention includes a flexible packaging bag made of multilayer composite aluminum foil or high-barrier plastic film, the packaging bag being divided into three independent sealed chambers, namely zone A, zone B and zone C. Zone A contains a concentrated solution, zone B contains a diluent, and zone C contains a detection substrate, the detection substrate including a separated background control zone.

7. The instant color-developing wipes as described in claim 6, characterized in that, Isolation seals are installed between adjacent areas of Zone A, Zone B and Zone C. The isolation seals are either easily bent and broken heat seals or weakly welded seals with micro-groove structures.

8. A method for protein detection using instant color-developing wipes as described in any one of claims 5-7, characterized in that, Includes the following steps: K.1 Gently bend along the isolation seals of areas A and B to break the isolation seals, allowing the concentrated functional liquid in area A to mix with the diluted liquid in area B. Shake gently several times to ensure even mixing, and the liquid will turn light brown. K.2 Squeeze one side of the mixed A / B area to allow the liquid pressure to break through the isolation seal of the C area. The mixed working liquid flows into the C area through the micro-groove. Let it stand for 5-10 seconds to ensure that the test substrate is fully and evenly wetted except for the background control area. K.3 Tear open the packaging bag along the easy-tear opening, take out the soaked test substrate, pinch the discolored area of ​​the test substrate or wipe the surface to be tested directly with a non-woven cloth. K.4 Observe the color change of the test substrate within 30 seconds. Compare the color of the wet discolored area with the color of the unwetted background control area of ​​a corner of the test substrate or with the provided standard color chart. If the wet wipe remains a light brown similar to the background control area or shows no obvious change, it indicates that the protein residue is below the detection limit. If a bright blue color is visible to the naked eye, it indicates that there is protein residue on the surface. Based on the depth of the blue, further semi-quantitative analysis can be performed.

9. The method as described in claim 8, characterized in that, The semi-quantitative method is as follows: compare with the control colorimetric card: if the color is light blue, the protein content is 1-5 μg; if the color is bright blue, the protein content is 5-50 μg; if the color is dark blue, the protein content is greater than 50 μg.

10. The use of the instant color-developing wipes as described in any one of claims 5-7 or the method as described in any one of claims 8-9 in the detection of proteins.

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