CN108036983B

CN108036983B - Method and instrument package for detecting metal ions in protein-containing sample

Info

Publication number: CN108036983B
Application number: CN201711296012.3A
Authority: CN
Inventors: 张永勇
Original assignee: Weiliyun Technology Beijing Co ltd
Current assignee: Shanxi Yunjincheng Culture Investment Co ltd
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2021-07-13
Anticipated expiration: 2037-12-08
Also published as: CN108036983A

Abstract

The invention discloses a method for detecting metal ions in a sample containing protein, which comprises the following steps: 1) adding a protein degrading enzyme to a sample containing protein to obtain an enzyme degradation product; 2) adding an acid to the enzymatic degradation product to obtain a mixture; 3) filtering the mixture to obtain a supernatant; 4) extracting the supernatant with an organic solvent, removing by-products soluble in the organic solvent to obtain an aqueous layer, washing the aqueous layer, and then detecting metal ions in the washed aqueous layer. The method has the advantages of simple process, no need of any professional laboratory instrument, high efficiency, rapidness and on-site detection. The method is suitable for detecting heavy metal ions in complex products such as milk. The invention also discloses an instrument package comprising reagents for carrying out the method.

Description

Method and instrument package for detecting metal ions in protein-containing sample

Technical Field

The invention relates to the technical field of metal ion separation and detection, in particular to a method and an instrument package for detecting metal ions in a sample containing protein.

Background

Metals (present as chemical moieties or ions), including heavy metals, are contaminants that are of increasing concern because of their potentially toxic, lethal impact on life systems and overall health. One way of contacting such metals is through food, in particular dairy products such as milk. In consideration of the continuous occurrence of contamination of dairy products in various regions of the world, it is imperative to detect and monitor metals.

Existing techniques for detecting metals, particularly heavy metals, are expensive, time consuming or use large (or cumbersome) equipment that requires the use of specialized laboratories, and therefore, prior to this application, field inspections were not achievable.

Milk is a typical example of a food product where metal detection is a concern. Milk is an emulsion or colloid of milk fat globules in an aqueous solution. The exact composition of the starting milk may vary, but generally it contains considerable concentrations of lactose, fat, protein and minerals as well as vitamins. Since field assay devices typically rely on biomolecular assays that employ DNA, RNA, or proteins, compositional determinations are an important issue for field assay detection techniques. The complex matrix of milk can interfere considerably and the preparation of the sample to be tested can take a considerable amount of time.

Therefore, in order to avoid the above drawbacks, there is a need to provide improved methods for separating metal ions from complex protein-containing substrates (e.g., milk) and solutions for detecting metal ions. The present disclosure provides new approaches to solving these and related problems.

Disclosure of Invention

The invention aims to provide a method for detecting metal ions in a sample containing protein, which has the advantages of simple process, no need of any professional laboratory instrument, high efficiency and high speed, and the detection time is 45-60 minutes.

In order to achieve the above object, a first aspect of the present invention provides a method for detecting metal ions in a protein-containing sample, the method comprising the steps of: 1) adding a protein degrading enzyme to a sample containing protein to obtain an enzyme degradation product; 2) adding an acid to the enzymatic degradation product to obtain a mixture; 3) filtering the mixture to obtain a supernatant; 4) extracting the supernatant with an organic solvent, removing by-products soluble in the organic solvent to obtain an aqueous layer, washing the aqueous layer, and then detecting metal ions in the washed aqueous layer.

The method disclosed in the present invention is general and has been demonstrated in the present application, for example, to be used for the detection of two different heavy metal ions (lead and uranium) in milk. The separated metal ions can be quantitatively detected using a bioassay method. The bioassay method used for the actual detection step is not limited. For example, conventional fluorescence detection methods or giant magnetoresistance effect (GMR) platform technology disclosed in the application publication No. US 2016011182A1, the entire contents of which are incorporated herein by reference in their entirety, may be employed. Other detection methods include colorimetric assays (e.g., using horseradish peroxidase), chemiluminescent, or electrochemical methods.

Depending on the nature of the protein-containing sample, any step in the method may be omitted. For example, the step of adding an acid to the enzymatic degradation product may be omitted. While this step is very useful when the protein-containing sample is milk, and may be used as an aid to break the milk, other samples may not be so treated because they may not be the starting emulsion, or the protein degrading enzymes may be sufficient to break down the protein-containing sample so that it can break the milk on its own. It is also possible to dispense with the step of using a protein-degrading enzyme, and it is sufficient to treat the protein-containing sample with a strong acid such as concentrated nitric acid. Thus, in certain embodiments, methods are provided that do not employ protein degrading enzymes. In certain such embodiments, the method does not employ proteinase K. In other embodiments, the method does not employ a strong acid.

In certain embodiments, the protein-containing sample is liquid milk. In other embodiments, the sample containing protein is milk powder. In a further embodiment, the protein-containing sample is a solution of protein powder. Thus, the disclosed methods can be used to detect metals, including heavy metals, in a variety of consumable products, including but not limited to protein supplements and related nutritional foods. Still further, the methods disclosed herein are particularly useful for detecting metals in dairy products including, but not limited to, butter, milk, cheese, cream, yogurt, sour cream, whey products, condensed milk, buttermilk, infant formula, milk protein concentrates, milk hydrolysates, and caseinates. The sample containing protein is not limited to dairy products, and the method disclosed by the invention can be used for detecting metals in plant and nut proteins, including proteins derived from soybean, almond, hazelnut, cashew and the like.

Further, the protein degrading enzyme employed in step 1) is proteinase K.

The main biomolecular components in milk and dairy-like products are proteins and fats. They are substances that interfere with most biological assays. To remove proteins, the present invention employs protein digesting enzymes. Further, proteinase K is preferred as a protein degrading enzyme because proteinase K is active under many different conditions and can function at room temperature without the need for any special buffers, which is also a commonly used enzyme in biochemistry, especially in cases where it is necessary to remove proteins without altering DNA or RNA (e.g.extracting chromosomal DNA from bacterial cells). Proteinase K digestion times also vary, with a minimum time of about 15 minutes required for protein digestion in the presence of about 90 units of proteinase K in milk at room temperature.

Other protein degrading enzymes may be used alone or in combination with proteinase K, including but not limited to serine proteases, threonine proteases, cysteine proteases, aspartic proteases, and glutamic proteases. Other protein degrading enzymes include, but are not limited to, various digestive enzymes such as pepsin, trypsin, chymotrypsin, metalloproteases, and elastase.

Further, the step 1) further comprises a denaturant. In the step of performing initial protein degradation using a protein degrading enzyme, a denaturing agent may preferably be added. Without being bound by theory, the denaturant may alter the spatial structure of the protein, facilitating entry of the protein degrading enzyme.

In some embodiments, the denaturing agent is one of a non-ionic surfactant, urea, a chelating agent, a thiol agent, a serine protease, or a combination thereof.

The surfactant includes anionic surfactants and nonionic surfactants. Wherein the anionic surfactant comprises one or more of ammonium dodecyl sulfate, potassium lauryl sulfate, sodium alkyl sulfate, sodium dodecyl benzene sulfonate and sodium stearate. The nonionic surfactant includes tween-20.

The total time spent by the process detection method disclosed by the invention is about 45-60 minutes, but the detection time can be shortened by adopting an additional reagent such as an anionic surfactant to improve the enzyme degradation rate.

Further, the acid used in step 2) is concentrated nitric acid. In other embodiments, the acid is hydrochloric acid or an acid increasing agent such as sodium acetate. The concentration of acid may be about 1M and the concentration of acid enhancer may be about 3M.

Further, a filter with the pore size of 3-5 microns is adopted in the step 3) to complete the filtering step. In certain embodiments, the filtration step can be accomplished using a filter having a pore size of 0.2 microns, although a pore size range of 0.05 to 1 micron can be very useful. In certain embodiments, the filter may be a nitrocellulose filter or a high efficiency protein binding filter membrane (e.g., a silica membrane).

Further, the detection step is completed using fluorescence detection in step 4).

Further, the detecting step is performed using a giant magnetoresistance measurement method in step 4).

In certain embodiments, the detecting step is accomplished using a colorimetric assay or an electrochemical sensor method. The following example illustrates the feasibility of the detection method.

US 2016011182a1 describes a giant magnetoresistance measurement method using a magnetic sensor in which one or more layers of the magnetic sensor are formed on a substrate so as to detect a magnetic field generated by magnetic nanoparticles brought into close proximity thereto, first ends of a first set of strands (design DNA or RNA having selectivity for detecting metal ions) are immobilized with respect to the magnetic sensor, and magnetic nanoparticles are attached to second ends of each strand in the first set of strands. When a sample containing metal ions is brought into contact with the substrate, the metal ions break at least a portion of the first set of chains, thereby causing the magnetic nanoparticles attached to the second ends of the broken chains to no longer be in proximity to the magnetic sensor, such that a corresponding interface measurement value of the detection device changes. In certain embodiments, other conventional giant magnetoresistance measurements may be used to detect the substrate.

Further, the invention provides a method for detecting heavy metal ions in a sample containing protein when the sample is a milk sample, wherein the method comprises the following steps: 1) adding protease K into a milk sample to obtain an enzyme degradation product; 2) adding nitric acid into the enzymatic degradation product to obtain a mixture after demulsification; 3) filtering the demulsified mixture to obtain a supernatant; 4) extracting the supernatant with chloroform, removing by-products soluble in chloroform to obtain an aqueous layer, washing the aqueous layer, and detecting heavy metal ions in the washed aqueous layer.

Fig. 1 shows a schematic diagram of a method for detecting heavy metal ions in a milk sample. In step 10, the sample is incubated with proteinase K to degrade the protein. In step 20, nitric acid (concentrated nitric acid) is added to demulsify and further denature the enzymatic degradation products obtained in the previous step. In step 30, the resulting mixture is filtered using a filter, such as a nitrocellulose filter. In step 40, chloroform or other suitable organic solvent is added and mixed thoroughly. In step 50, the aqueous layer is separated. In step 60, the aqueous layer is assayed to detect the presence of heavy metal ions by any means recited herein including fluorescence detection or giant magnetoresistance measurement.

Further, the heavy metal ion is lead. Further, the heavy metal ion is uranium. In certain embodiments, the methods in the case of detecting heavy metals in milk are directed to selectively detecting heavy metal ions. In certain embodiments, the detection method is directed to detecting two or more heavy metal ions, such as two, three, four, or even five heavy metal ions, simultaneously.

A second aspect of the invention provides an instrument package comprising: a container for holding a sample containing protein; proteinase K; nitric acid; chloroform and instructions for separating metal ions from a sample containing proteins.

Further, the instrument package is a small instrument package for separating metal ions in situ.

Further, the kit further comprises a handheld detection device for detecting metal ions.

Further, the hand-held detection device adopts a fluorescence detection method or a detection method based on giant magneto-resistance effect (GMR).

Further, the kit further comprises a protein denaturing agent. The protein denaturing agent comprises one or a combination of Sodium Dodecyl Sulfate (SDS), urea, ethylene diamine tetraacetic acid, trypsin and chymotrypsin. The kit may also include necessary buffers, small glass vials of deionized water, and other reagents.

The invention has the following advantages:

the invention aims to provide a method for detecting metal ions in a protein-containing sample in situ, which has simple process, avoids using any special laboratory instrument, and has high efficiency and rapidness.

Drawings

Fig. 1 shows a schematic diagram of a method for detecting heavy metal ions in a milk sample.

FIG. 2 shows the effectiveness of acid on separating lead ions from a milk sample, where the filled circles indicate the fluorescence signal of the DNase-type sensor detected in the absence of lead and the open circles indicate the presence of 500nM Pb in the presence of Pb²⁺In this case, the detected fluorescence signal, the result in water, is used as a control to evaluate the effectiveness of the acid demulsification protocol.

FIG. 3 shows the effectiveness of the type of filtration membrane for separating lead ions from a milk sample, the Pb present in which could not be detected using regenerated nitrocellulose (pore size 0.2 μm)²⁺。

FIG. 4 shows the effectiveness of the filtration method for separating lead ions from a milk sample, the filled circles indicating the fluorescence signal detected in the absence of lead and the open circles indicating the presence of 500nM Pb²⁺The fluorescence signal detected in the case of (a), the results in water, was used as a control to evaluate the effectiveness of the filtration method.

FIG. 5 shows the effectiveness of organic solvents in separating lead ions from milk samples, the filled circles indicating the fluorescence signal detected in the absence of lead, and the open circles indicating the presence of 500nM Pb²⁺The fluorescent signal detected in the case of (1), Phe-CHCl₃IAA is a mixture of phenol, chloroform and isoamyl alcohol, CHCl₃The results in water were used as a control for chloroform to evaluate the effectiveness of the extraction protocol.

Figure 6 shows the effectiveness of proteinase K on the separation of heavy metal ions from milk samples.

FIG. 7A shows the fluorescence signal detected in the aqueous layer separated from the milk sample together with Pb²⁺And a line graph of the ion concentration relationship shows that the lead ions can be quantitatively detected.

FIG. 7B shows the fluorescence signal detected in the aqueous layer separated from the milk sample together with the UO₂ ²⁺And a line graph of the relationship of the ion concentration shows that the uranium ions can be quantitatively detected.

FIG. 8A shows the GMR signal detected in the water layer separated from the milk sample together with Pb²⁺And a line graph of the ion concentration relationship shows that the lead ions can be quantitatively detected.

FIG. 8B shows the detected GMR signal from the water layer separated from the milk sample and the UO₂ ²⁺And a line graph of the relationship of the ion concentration shows that the uranium ions can be quantitatively detected.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The components and percentages in the following examples are by weight unless otherwise indicated. The room temperature is 20-25 ℃.

Example 1

This example describes an exemplary method for separating heavy metal ions from a milk sample and detecting the heavy metal ions using a fluorescence detection method with a magnetic sensor.

Selection of acid

To optimize the detection of heavy metal ions in milk samples, acids that break the enzymatic degradation products were tested, including nitric acid (1M), hydrochloric acid (1M), and low pH sodium acetate buffer (3M, pH 5.2), all tested at 167.5 ml. The results show that Pb can be detected using both acid and sodium acetate buffers²⁺Is present. FIG. 2 shows the effectiveness of acid on separating lead ions from a milk sample, where the filled circles indicate the detected fluorescence signal of the DNase-type sensor in the absence of lead and the open circles indicate the presence of 500nM Pb in the presence of Pb²⁺The detected fluorescence signal in the case of (2). As a control, the effectiveness of the acid demulsification scheme was assessed by measuring the fluorescence signal detected by the sample water before and after addition of the heavy metal ions. Nitric acid showed the best results compared to water, which served as a control. Thus, for further experiments, nitric acid was used. Predicted sulfurAcids will also play the same role in this step.

Filter selection

The purpose of filtration is to remove proteins and fats, so the choice of filtration membrane is limited to polyvinylidene fluoride (PVDF) and nitrocellulose. Both of the above filtration membranes have high protein binding ability and have various pore sizes. Nitrocellulose is suitable for binding low molecular weight proteins. Since proteinase K breaks down most proteins into smaller fragments, efficient binding to low molecular weight proteins is considered desirable in this case. Among the various pore sizes suitable, a pore size of 0.2 μm is mainly selected, which can prevent the small peptide fragments from passing through without clogging the filter. In addition, regenerated nitrocellulose (pore size of 0.2 μm) was used, but heavy metal ions could not be detected after sample preparation, and we considered that the main reason for this was the low protein-binding ability of regenerated nitrocellulose. Figure 3 shows the effectiveness of a filter membrane type for separating lead ions from a milk sample. Failure to detect the presence of Pb in milk samples using a regenerative nitrocellulose filter²⁺。

As an alternative method to nitrocellulose, polymethyl methacrylate beads and silica beads, each of which has the ability to absorb proteins located on the surface thereof, are further used. However, after separation by beads, the signals detected indicate that they are not efficient at removing protein and fat. Figure 4 shows the effectiveness of the filtration method for separating lead ions from a milk sample. Filled circles indicate the fluorescence signal detected in the absence of lead, open circles indicate the presence of 500nM Pb²⁺The detected fluorescence signal in the case of (2). As noted above, the results in water were used as a control to evaluate the effectiveness of the filtration process.

Selection of organic solvent for extraction

Even after demulsifying the milk and filtering using a nitrocellulose filter, residual protein and fat are expected to remain in the filtrate. In biochemistry, organic solvents are often used in order to remove trace amounts of proteins from nucleic acid solutions. A classical example is the extraction of DNA or RNA after the enzymatic process using phenol chloroform. In this method, proteins are denatured in an organic solvent and settled in the interface between an aqueous layer and an organic layer. Salts and nucleic acids still remain in the aqueous layer. Since fats are soluble in non-polar organic solvents, organic solvents that can be used to remove proteins and fats include, but are not limited to, chloroform, ethyl acetate, and mixtures of phenol, chloroform, and isoamyl alcohol, provided that organic solvent extraction also removes fats.

Figure 5 shows the effectiveness of organic solvents in separating lead ions from milk samples. Filled circles indicate the fluorescence signal detected in the absence of lead, open circles indicate the presence of 500nM Pb²⁺The detected fluorescence signal in the case of (2). Phe-CHCl₃IAA is a mixture of phenol, chloroform and isoamyl alcohol, CHCl₃Is chloroform. As in the acid-selective optimization process, the results in water were used as a control to evaluate the effectiveness of the extraction protocol.

For a mixture of phenol, chloroform and isoamyl alcohol, centrifugation is necessary to achieve two-phase separation of the organic layer and the aqueous layer. In addition, the results show that the activity is not optimal after isolation. For ethyl acetate and chloroform, phase separation was achieved without performing centrifugation. Both chloroform and ethyl acetate showed the expected level of activity. When the supernatant was extracted with chloroform, chloroform was identified as the most user-friendly organic solvent since the aqueous layer was the upper layer and was easily separated. For ethyl acetate, the aqueous layer is the bottom layer, so if the operator is not expert, then removing ethyl acetate may require extreme skill.

Additional method combinations

In order to simplify the process, it is systematically evaluated whether a step is necessary for the entire process by omitting it one step at a time.

First, the enzymatic step of proteinase K was omitted from the process and 167.5. mu.L of nitric acid (1M) was added to the diluted milk sample. Sequentially filtering with nitrocellulose filter membrane and extracting with chloroform to obtain waterLayer, detection of the presence of Pb in the sample²⁺Ions. However, the activity was found to be suboptimal when compared to control sample water. Figure 6 shows the effectiveness of proteinase K on the separation of heavy metal ions from milk samples.

Additionally, the omission of a nitrocellulose filter membrane from the process was evaluated to determine whether the simple use of organic solvent extraction was sufficient to remove protein and fat. Chloroform is used as the organic solvent, and a large amount of protein still remains in the interface of the organic phase and the water phase in the extraction process. Efficient extraction of the aqueous layer is therefore critical, and at least five successive extraction steps must be carried out before the separation of the two phases without disturbing the interface, which significantly increases the complexity of the process and, in addition, it has been found that the activity of the sensor is not optimal. This indicates that without the use of nitrocellulose filter membranes, simple extraction with organic solvents is not sufficient to completely remove proteins and fats.

The process does not depend on the detection method

Once the heavy metal has been separated from the milk sample, a dnase-type sensor specific for a single metal ion can be used to detect the heavy metal ion. The detection method of heavy metal ions can be realized using a fluorescent dye (Cy3, Cy5, FAM, etc.) or by a giant magnetoresistance effect (GMR) method (i.e., magnetic nanoparticles). All results show that the above results can be obtained using the fluorescent stain Cy 3. However, similar results can be obtained using the giant magnetoresistance effect (GMR) method shown below.

1mL of whole milk was diluted to 5mL using deionized water in a glass or plastic tube. mu.L proteinase K (0.8U/mL, New England Biolabs, Ipusvie, Mass.) was added to the milk and incubated at room temperature for 15 minutes. After 15 minutes of incubation, 167.5 μ L of 1M nitric acid was added to the tube and mixed well to break the milk. A1 mL aliquot of this demulsified milk sample was removed using a 1mL syringe and passed through a nitrocellulose filter (pore size 0.2 μm, Maine Engineering). Subsequent detection of metal ions is dependent on efficient filtration, and thus any white suspension in the filtrate indicates that the nitrocellulose filter is not efficient at removing protein and fat. In this case, the filtering step is re-executed. The clear filtrate is collected in a centrifuge tube with the volume of 1.5mL, and the total volume of the filtrate is 200-400 mu L, which is sufficient for the subsequent DNA enzyme type determination method.

After filtration, an equal amount of chloroform was added to the filtrate and mixed well, and the tube was left alone for 5 minutes. The uppermost aqueous layer was carefully removed using a pipette and the aqueous layer was placed into a new 0.5mL centrifuge tube. In the functional DNA type assay method, a sample volume of 50. mu.L is sufficient to determine the presence or absence of metal ions. FIG. 7A shows the fluorescence signal detected in the aqueous layer separated from the milk sample together with Pb²⁺Line graphs of ion concentration showing quantitative detection of lead ions, FIG. 7B shows fluorescence signal detected from water layer separated from milk sample and UO₂ ²⁺The line graph of the relationship of the ion concentration shows that uranium ions can be detected quantitatively, and no peak value change of the heavy metal ion solution occurs in the detection result, so that the conclusion that no heavy metal ions exist in the sample can be obtained under the condition.

Detection of heavy metals using DNase-type assays

DNA microarrays are constructed by immobilizing matrix DNA on slides containing Codelink (TM) surfaces (Surmodics, Ildenpril, Minn.). The substrate was dissolved in an imprinting buffer containing sodium phosphate and polyvinyl alcohol and spotted onto a slide using a spotting instrument with a distance of 400 microns between adjacent spot centers (science AG, berlin, germany) and adsorption was accomplished in a sealed cabinet at a temperature of about 18 ℃ and a humidity of about 70%.

After adsorption, the slides were placed in a humidity chamber at room temperature for about 12-16 hours. The substrate was immobilized on the slide by reaction between NHS lipid groups on the surface of the cotelink tm and primary amine at the 3' -end of the substrate. And (3) blocking redundant functional groups on the glass slide by using a 50mM ethanolamine solution, washing the glass slide by using deionized water after blocking redundant functional groups on the glass slide by using the ethanolamine solution, and carrying out rotary drying to obtain the gene chip, wherein each glass slide is distributed with twelve subarrays, and polystyrene is arranged around each subarray for separating the subarrays. This process allows 12 reaction chambers to be created per slide.

The DNase sensor which is 1000 times more than the solidified substrate is dissolved in the reaction buffer solution, 85 mu L of DNase sensor solution and 0.85 mu L of heavy metal ion solution are respectively added into the reaction cavity for reaction, and the reaction time is 15 minutes. The solution was then removed from the reaction chamber by pipette, and the reaction chamber was rinsed twice with 85 μ L of reaction buffer to remove residual lysed substrate and excess dnase-type sensor.

A step of detecting metal ions by a fluorescence method: mu.L of Cy3 streptavidin stain (5 ug/mL in reaction buffer) was added to the reaction chamber and reacted for 30 min at room temperature. After the stain was cleared, the slides were rinsed with 0.5% sodium dodecyl sulfate solution and deionized water. Slides were photographed using an Axon GenePix 4000B biochip scanner (Axon Instruments, foster city, ca) with a Cy5/Cy3 filter at a resolution of 5 μm. The laser power and photomultiplier tube voltage (PMT) are set to obtain the optimum signal intensity. The original 16-bit tiff format photographs were quantified using GenePix Pro 6.0(Axon Instruments, foster, ca).

Detecting metal ions by using a giant magnetoresistance method: 80 μ L of streptavidin coated magnetic nanoparticles were added to the reaction chamber and the signal detected and recorded using a giant magnetoresistance device. FIG. 8A shows GMR signal detected with a giant magnetoresistive sensor and Pb²⁺And a line graph of the ion concentration relationship shows that the lead ions can be quantitatively detected. FIG. 8B shows GMR signal detected with a giant magnetoresistive sensor versus UO₂ ²⁺And a line graph of the relationship of the ion concentration shows that the uranium ions can be quantitatively detected.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for detecting metal ions in a protein-containing sample, wherein the protein-containing sample is a milk sample, the method comprising the steps of:

1) adding protease K into a milk sample to obtain an enzyme degradation product;

2) adding nitric acid into the enzymatic degradation product to obtain a mixture after demulsification;

3) filtering the demulsified mixture to obtain a supernatant, and finishing the filtering step by adopting a filter with the aperture of 3-5 microns in the step 3);

4) extracting the supernatant by using chloroform, removing a byproduct soluble in the chloroform to obtain a water layer, then flushing the water layer, and detecting heavy metal ions in the flushed water layer, wherein the heavy metal ions are lead or uranium; the detection step is accomplished in step 4) using fluorescence detection or using giant magnetoresistance measurements.

2. The method for detecting metal ions in a protein-containing sample according to claim 1, further comprising using a denaturing agent in step 1), wherein the denaturing agent is one of an anionic surfactant, urea, a chelating agent, a thiol reagent, and serine protease, or a combination thereof.

3. An instrument pack, comprising:

a container for holding a sample containing protein;

proteinase K;

nitric acid;

chloroform and instructions for separating metal ions from a sample containing proteins.

4. The kit of claim 3, wherein the kit is a small kit for isolating metal ions in situ.

5. The kit of claim 3, further comprising a handheld detection device for detecting metal ions, wherein the handheld detection device employs fluorescence detection or giant magnetoresistance effect (GMR) based detection.

6. The kit of claim 3, further comprising a protein denaturing agent comprising one or a combination of Sodium Dodecyl Sulfate (SDS), urea, ethylenediaminetetraacetic acid, trypsin, and chymotrypsin.