CN102417925A - Reagent for detecting content of potassium ions in blood and preparation method thereof as well as kit containing same - Google Patents

Abstract

The invention relates to a reagent for detecting content of potassium ions in blood and a preparation method thereof as well as a kit containing the same. The reagent comprises nucleic acid molecules, heme and a luminol reagent, wherein, the nucleic acid molecules are guanosine-enriched oligonucleotides which can form a quadruplex structure, and the sequential formula is R-Gn-Nm-Gn-Nm-Gn-Nm-Gn-R'. The preparation method comprises the following steps of: (1) evenly mixing polyvinyl alcohol-phenyl ethylene pyridine hydrosol, the nucleic acid molecules, the heme and the luminol reagent to form a sol status; and (2) in the case of ultraviolet irradiation on the mixture obtained in step (1), cross-linking phenyl ethylene pyridine so that the mixture is cured and the nucleic acid molecules, the heme and the luminol reagent are physically fixed in gel so as to form a detection layer. The preparation method provided by the invention integrates advantages of the existing blood potassium ion detection methods, and has the characteristics of high sensitivity, high selectivity and low cost, and is simple in operation and convenient in use.

Description

A kind of test kit that detects the reagent and the preparation method of potassium content in the blood and contain this reagent

Technical field

The invention belongs to the detection kit field, especially a kind of test kit that detects the reagent and the preparation method of potassium content in the blood and contain this reagent.

Background technology

Potassium is the human body trace elements necessary, accounts for 0.2% of body weight for humans.Potassium element plays a very important role keeping on the human body normal physiological function.Potassium is to regulate the inside and outside osmotic pressure equilibrated important element of cell; Also be to keep the requisite element of nerve system of human body function; Potassium ion passes through the turnover potassium-channel, the potential balance of cell membrane, and very important effect is played in the conduction of nerve signal and muscle control.As hydrionic replacement, also play crucial effect in the acid of potassium ion inside and outside cell.In addition, potassium ion has been participated in Biochemical processes important in the human body, and for example Triphosaden (ATP) is synthetic, glycogen synthetic, the secretion of some mineralocorticoid such as aldosterone etc.Potassium ion can also through to telomere (telomere) thus the regulation and control pair cell apoptosis of structure and cancer form the generation great effect.

The intravital potassium element of people exists with ionic species; Overwhelming majority potassium ion is present in enchylema and the blood.In blood the normal concentration scope of potassium 3.5 to 5mEq/L; And the concentration of potassium is about 140mEq/L in enchylema.If the potassium concentration in the blood (blood potassium) is lower than normal range, will produce a series ofly such as symptoms such as diarrhoea, vomiting, muscle weakness and irregular pulse thereof, then can produce greatly infringement for a long time to renal function in the past.If blood potassium is higher than normal range, symptoms such as fatigue, arrhythmia and atrial fibrillation are arranged then.The unusual symptom of blood potassium level does not have specificity, so clinical diagnosis blood potassium must be measured the blood potassium level unusually.

The measurement of blood potassium at present belongs to conventional sense, and detection means mainly contains atomic absorption method, ion selective electrode method, enzyme process, fluorimetry etc.Detection method relatively see table 1.

The comparison of the existing blood potassium detection method of table 1

The highly sensitive selectivity of atomic absorption method is good, but complicated operation.Fluorimetry and ion selective electrode method are easy and simple to handle, but relatively poor to the selectivity of sodium.Enzyme process costs an arm and a leg and complicated operation.

Thymus nucleic acid (DNA) has the effect of catalysis biochemistry even organic reaction in some fragments of the nineties discovery in last century DNA except having the function of preserving genetic information.These dna fragmentations with katalysis are become nucleicacidase (DNAzyme).All nucleicacidases all obtain through artificial selection (in vitro selection) in the laboratory at present.In the process of artificial selection,, can make the nucleicacidase of artificial selection have fabulous selectivity and the catalytic activity that meets demand through artificial control reaction conditions.

Biochemical analysis is the present most important applications of nucleicacidase.Nucleicacidase is applied to metals ion for example zinc, lead, copper and uranium, and the detection of specific nucleic acid sequence.Biosensor based on nucleicacidase generally has very high selectivity and satisfactory sensitivity; Compare with chemical sensor, have the strong characteristics of good water solubility and interference resistance, still; The poor stability of nucleicacidase in the aqueous solution; Fluorescently-labeled nucleic acid molecule costs an arm and a leg, so the application of nucleicacidase in biochemical analysis obtains restriction, present research situation also has bigger gap from practical application.

Summary of the invention

The objective of the invention is to overcome the weak point of prior art, the reagent and the preparation method of potassium content in a kind of selectivity detection blood high, highly sensitive and easy to use is provided and contains the test kit of this reagent.

The objective of the invention is to realize through following technical scheme:

A kind of reagent that detects potassium content in the blood; Comprise nucleic acid molecule, protoheme and luminol reagent; Said nucleic acid molecule is for forming the oligonucleotide that is imbued with guanosine of four chain body structures, and the sequence formula is R-Gn-Nm-Gn-Nm-Gn-Nm-Gn-R ', and wherein m, n are integer.

And the mol ratio of said nucleic acid molecule and protoheme and luminol reagent is 1: 10: 1000.

And said nucleic acid molecule is following:

Sequence
	5′-GGG-ATT-GGG-ATT-GGG-ATT-GGG
5′-GGGG-ATT-GGGG-ATT-GGGG-ATT-GGGG
	5′-GGGGG-ATT-GGGGG-ATT-GGGGG-ATT-GGGGG
5′-TAATGGGG-ATT-GGGG-ATT-GGGG-ATT-GGGG-TAAT
	5′-ACTTAATGGGG-ATT-GGGG-ATT-GGGG-ATT-GGGGACTTAAT

A kind of preparation method who detects the reagent of potassium content in the blood comprises the steps:

(1) Z 150PH-water-sol of phenyl vinylpyridine, nucleic acid molecule, protoheme, luminol reagent are mixed the back and become collosol state;

(2) with the mixture of step (1) by uv irradiating, the phenyl vinylpyridine produces crosslinked, makes mixture solidified, nucleic acid molecule, protoheme, luminol reagent in gel, are formed detection layers by physical fixation;

The mol ratio of the water-sol of said Z 150PH-phenyl vinylpyridine, nucleic acid molecule, protoheme, luminol reagent is 1: 10: 1000.

And, being shaped on the hydrogel supporting layer in addition in the lower floor of detection layers, composition is the linear polymer of polyoxyethylene glycol and polyesteramide, is shaped on the carbon black resist on the upper strata of detection layers.

And concrete preparation process is following:

(1) dissolving nucleic acid molecule and protoheme in the HEPES of 50mM buffered soln pH 7.0 are heated to 80 ℃ with the solution with water bath and also are cooled to room temperature at normal temperatures so that the molecular structure homogenization of nucleic acid adds luminol reagent in solution;

(2) on plastics film, be coated with the film of last layer D4 hydrogel and make it natural air drying with the scraper film applicator, thickness is 90-110 μ m;

(3) in 10wt%PVA-Sbq solution, add the solution of step (1) preparation and stirring,

(4) on the film of the D4 hydrogel of step (2), be coated with last layer step (3) and obtain solution, uv lamp is placed top 25-35 minute of rete, make phenylpyridine ethene crosslinked and rete is become dry;

(5) the last the superiors are coated with last layer with the scraper film applicator and contain 10wt% sooty D6 hydrogel and make the rete natural air drying.

A kind of test kit that detects the reagent of potassium content in the blood that contains.

Advantage of the present invention and beneficial effect are:

1, the present invention has prepared the detection that domestic first transmitter based on nucleicacidase is used for clinical blood potassium; Nucleicacidase is fixed on the solid substrate; And thoroughly solve the storage and transport problem of nucleicacidase transmitter; The nucleicacidase that this transmitter uses has high selectivity to potassium ion, with making the transmitter of research and development have with the similar selectivity of flame optical spectroscopy, and with the ease of similar sensitivity of other analytical procedures and use.

2, the method that the application invented has highly sensitive and highly selective and simple to operate, easy to use and cheap characteristics with the advantage of integrated each existing method.

3, the application will be fixed on test kit on the test kit the responsive nucleicacidase of potassium ion; And be to detect behind the fluorescent signal with the catalytic reaction conversion of nucleicacidase; This test kit can detect the above potassium of 0.2mM; And have very high selectivity, sodium ion will not disturb detecting to produce in blood.

Description of drawings

Fig. 1 is the principle of blood potassium ion transducer/test kit of the present invention;

Fig. 2 is the crosslinking reaction formula of phenyl vinylpyridine of the present invention;

Fig. 3 is the structure of the test kit of blood potassium ion transducer use of the present invention;

Fig. 4 is the fluorescence response in time of the present invention's sample of test kit under different potassium concentrations;

Fig. 5 gain in strength for the fluorescence that calculates according to Fig. 4 and potassium concentration between dependency.

Embodiment

Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.

If the per-cent that following examples are mentioned does not have special indicating all to be weight percentage, the mM of unit representes every liter of mmole.

One, the ultimate principle of blood potassium ion transducer

Blood potassium ion transducer is based on the responsive nucleicacidase of potassium ion is designed.

This nucleicacidase comprises three parts:

1, one section oligonucleotide that is imbued with guanosine (dna fragmentation) that can form four chain body structures;

2, protoheme;

3, promote the potassium ion that four chain body structures form.

Its catalytic mechanism is as shown in Figure 2, if lack potassium ion in the sample, the structure of oligonucleotide in solution that is imbued with guanosine will be at random with uncertain.If contain potassium ion in the sample, potassium ion and oligonucleotide interact and folding this section oligonucleotide, form four chain body structures.Four chain body structures are laminate structure (shown in Fig. 1 grey dashed square), verified can the interaction with bigger planar molecule, make planar molecule can be embedded in these laminate structures ( HanH, Langley DR, Rangan A, Hurley LH, J.Am.Chem.Soc., 123,8902-8013 (2001)).Haemachrome molecule is a two dimensional structure, also can be embedded in the laminate structure of four serobilas.Verified ( Han H, Langley DR, Rangan A, Hurley LH, J.Am.Chem.Soc., 123; 8902-8013 (2001)); When haemachrome molecule was embedded in the four serobila laminate structures, four serobilas-protoheme duplex had peroxidase activity, and the reaction of hydrogen peroxide oxidation luminol reagent is accelerated more than three one magnitude.Can produce photon in the oxidising process of luminol reagent and detected by instrument.As stated, because the generation of photon determines by forming of nucleicacidase, so the existence of potassium ion is necessity and essential to the detection of photon.Just can detect the existence of potassium ion thus through the generation that detects photon; And the concentration of speed that photon produces and nucleicacidase is relevant; Indirectly the concentration with potassium ion is relevant, thus the speed that produces through measurement of photon, concentration that just can the corresponding calculated potassium ion.

This Selectivity of Sensor is by the selectivity decision of four chain body structures.In blood sample because Na ion concentration is about the thirtyfold of potassium concentration, add that the height of potassium ion and sodium ion character and structure is similar, so the interference that sodium ion causes be present except the flame optical spectroscopy common issue of blood potassium detection method.Four chain body structures have high selectivity for potassium ion: if it is identical to form the ability of four chain body structures, the sodium ion desired concn is hundreds of times of potassium ion desired concn.Na ion concentration in the blood sample is far below this, so this transmitter will have high selectivity and very low background signal.

Two, nucleic acid molecule is fixing

In test kit, nucleic acid molecule will be fixed in the solid phase substrate, and concrete laminate structure is following:

The lowest layer is the hydrogel supporting layer, and staple is urethane-polyoxyethylene glycol hydrogel, (concrete structure is seen following patent: US2008/0152864A1) its mainly to act on be to isolate PVA-Sbq (Z 150PH-phenyl vinylpyridine) layer contain nucleicacidase.

Nucleicacidase comprises that nucleic acid molecule and protoheme and luminol reagent are fixed on the second layer, and the staple that plays fixed action is the multipolymer of Z 150PH and phenyl vinylpyridine.The water-sol of Z 150PH-phenyl vinylpyridine and nucleicacidase and luminol reagent are mixed to become collosol state after even.After mixture was by uv irradiating, the phenyl vinylpyridine produced crosslinked, makes mixture solidified; And nucleicacidase and luminol reagent also will be by physical fixation in gels.Its reaction formula is seen Fig. 2.

Because nucleicacidase is fixed in the solid and the preservation that is dried, so the hydrolysis of nucleic acid comparatively speaking can be difficult a lot: at first owing to lack of water, the possibility that hydrolysis takes place reduces greatly; Secondly owing to the existence of solid phase resist, nucleic acid lytic enzyme contact nucleic acid molecule is difficulty relatively, and resulting in its catalytic hydrolysis reaction can not take place.The protein enzyme that this principle has been fixed is confirmed, therefore can expect that the nucleicacidase that is fixed is also with more stable.Different with the protein enzyme, the folding of nucleicacidase is reversible, so the activity of the nucleicacidase of kept dry also is guaranteed.

Three, the making method of test kit:

1, the sequence of nucleicacidase:

The necessary coincidence formula R-Gn-Nm-Gn-Nm-Gn-Nm-Gn-R ' of the sequence of nucleicacidase amplifying nucleic acid molecule, spendable molecule is listed in table 2 (but being not limited only to table 2).

Table 2 can be used for the nucleicacidase sequence of blood potassium ion transducer

Title	Sequence
		G-3	5′-GGG-ATT-GGG-ATT-GGG-ATT-GGG
G-4	5′-GGGG-ATT-GGGG-ATT-GGGG-ATT-GGGG
		G-5	5′-GGGGG-ATT-GGGGG-ATT-GGGGG-ATT-GGGGG
G-4-R4	5′-TAATGGGG-ATT-GGGG-ATT-GGGG-ATT-GGGG-TAAT
		G-4-R7	5′-ACTTAATGGGG-ATT-GGGG-ATT-GGGG-ATT-GGGGACTTAAT

2, the preparation of the fixing and test kit of nucleicacidase

Dissolving nucleic acid molecule and protoheme in the HEPES of 50mM buffered soln (pH 7.0).The solution with water bath is heated to 80 ℃ also is cooled to room temperature at normal temperatures so that the molecular structure homogenization of nucleic acid adds luminol reagent in solution.This solution will be used for the immobilization of nucleicacidase.On a clean plastics film, (a kind of urethane-polyoxyethylene glycol hydrogel, specifically see following patent: film US 2008/0152864A1) also makes it natural air drying to be coated with last layer D4 hydrogel with the scraper film applicator.Nucleic acid/protoheme/luminol reagent the solution that in 10%PVA-Sbq (Z 150PH-phenylpyridine ethene complex compound) solution, prepares in the adding said process also stirs.Be coated with the PVA-Sbq solution of last layer nucleic acid with the scraper film applicator.Uv lamp is placed the top 30 minutes of rete, make phenylpyridine ethene crosslinked and rete is become dry.US 2008/0152864A1) and make the rete natural air drying being coated with last layer with the scraper film applicator in the above at last contains 10% sooty D6 hydrogel (a kind of urethane-polyoxyethylene glycol hydrogel is specifically seen following patent:.

Use cutting machine to extract diameter and be about the disk of 5mm and be installed in the sky test kit, the shape of last test kit is seen Fig. 3.

Blood sample gets into test kit and the valve through black through kapillary from the test kit right part and contacts with transmitter and produce signal and received by luminoscope.The position of having reserved other five other transmitters at present on the test kit has reached the purpose of a plurality of detection things of one-time detection.

Four, detect instance

1, test kit preparation process:

(1) in the HEPES of 50mM solution, adding nucleic acid molecule (G-4), to make the ultimate density of nucleic acid be 0.3mM, in this solution of 1mL, adds the 2mg protohemine;

(2) take out the solution of preparation in the step (1) is heated to 80 ℃ in water-bath after, and naturally cool to room temperature;

(3) in 2.6g contains 10% PVA-Sbq solution, add the HEPES solution (pH 7.0) of 0.7mL step (2) prepared solution, 37mg o-aminophthalylhydrazide and 0.3mL 500mM and lucifuge stirs on ice;

(4) on a dustless plastics film, being coated with last layer concentration is that 4wt%, thickness are D4 hydrogel rete and the natural air drying (about 30 minutes consuming time) of 100 μ m;

(5) coat the nucleic acid solution of PVA-Sbq prepared in the step (3) on the film that in step (4), prepares, the thickness of rete is 100 μ m, and film was placed uv lamp following 30 minutes;

(6) in step (5), be coated with solution and the natural air drying that last layer contains 6wt% carbon black and 1.8wt%D6 hydrogel on the prepared film;

(7) extract in the step (6) prepared film and be installed on the test kit and can measure.

2, sample measurement:

Containing 50mM HEPES, 150mM sodium-chlor adds hydrogen peroxide in the aqueous solution of 2.5mM calcium chloride and different concns Repone K, makes the hydrogen peroxide final concentration reach 10mM.

Drive the sample solution of about 150 μ L and assign on the test kit and begin measurement with kapillary two with portable luminoscope OPTI CCA-TS.

3, data analysis:

The computer that links to each other with luminoscope will write down the photo emissions intensity of reaction generation and the curve of time; Can be according to this curve in the hope of reacting preceding ten seconds speed of reaction; This speed is exactly the response of test kit to potassium ion, can be in the hope of the concentration of potassium ion according to typical curve.

4, experimental data:

Test kit is to the response of potassium ion:

Response shows at Fig. 4 the test kit that above-mentioned steps obtains to potassium ion.Fig. 4 is the fluorescence response in time of the sample of test kit under different potassium concentrations.Can see that under different potassium concentrations, the speed that fluorescence rises is different.When only having the potassium ion of 0.2mM in the solution, fluorescence intensity has risen about 4% in 60 seconds.And when containing the potassium ion of 2mM in the solution, fluorescence intensity has risen about 15% in 60 seconds.So the concentration of fluorescence enhanced speed and potassium ion is closely-related, the speed that also can use fluorescence to increase is calculated the concentration of potassium ion.

Fig. 5 represent according to the fluorescence that Fig. 4 calculates gain in strength and potassium concentration between dependency.Can more significantly see the dependency that potassium ion and fluorescence are advanced the speed.Under the lower situation of potassium concentration, potassium concentration and fluorescence are advanced the speed linear.In the scope of 0-10mM, can carry out match with the equation that index increases.The sample that does not contain potassium ion is compared, and it is big that the caused response of the potassium ion of 0.2mM obviously becomes, so the sensitivity of this test kit is below 0.2mM.

5, the selectivity of test kit:

Because alkalimetal ion comprises the high similarity of sodium ion and potassium ion, result in prior art and can not get rid of the interference that these pair ions detect fully.The selectivity of the test kit that therefore, is necessary the application is mentioned is measured.

The selectivity of test kit uses the concentration of identical needed other metals ions of signal that produce with the 1mM potassium ion to represent.The selectivity of this test kit is shown in 3 tables.

The selectivity of table 3 blood potassium test kit

Ion

Sodium

Lithium

Magnesium

Calcium

Zinc

Iron (divalence)

Selectivity (mM)

?＞500

?＞500

150

180

150

100

Since in blood above-mentioned ionic content all far below on shown in the table, so saying of can affirming, this test kit does not have the interference of other metallic cations when using blood sample.

6, conclusion

The application has set forth the test kit that a kind of novel being used for detects the blood potassium content.This test kit will be fixed on the test kit the responsive nucleicacidase of potassium ion, and the catalytic reaction conversion of nucleicacidase is fluorescent signal and is able to detect.This test kit can detect the above potassium of 0.2mM at an easy rate, and has very high selectivity, and sodium ion will not disturb detecting to produce in blood.

SEQUENCE?LISTING

 

< 110>the Tianjin Skien is thought biochemical technology ltd

 

< 120>a kind of test kit that detects the reagent and the preparation method of potassium content in the blood and contain this reagent

 

<130> 2011-10-13

 

<160> 5

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 21

<212> DNA

<213> G-3

 

<400> 1

gggattggga?ttgggattgg?g 21

 

 

<210> 2

<211> 25

<212> DNA

<213> G-4

 

<400> 2

ggggattggg?gattggggat?tgggg 25

 

 

<210> 3

<211> 29

<212> DNA

<213> G-5

 

<400> 3

gggggattgg?gggattgggg?gattggggg 29

 

 

<210> 4

<211> 33

<212> DNA

<213> G-4-R4

 

<400> 4

taatggggat?tggggattgg?ggattggggt?aat 33

 

 

<210> 5

<211> 39

<212> DNA

<213> G-4-R7

 

<400> 5

acttaatggg?gattggggat?tggggattgg?ggacttaat 39

 

 

Claims (7)

1. reagent that detects potassium content in the blood; It is characterized in that: comprise nucleic acid molecule, protoheme and luminol reagent; Said nucleic acid molecule is for forming the oligonucleotide that is imbued with guanosine of four chain body structures; The sequence formula is R-Gn-Nm-Gn-Nm-Gn-Nm-Gn-R ', and wherein m, n are integer.

2. the reagent of potassium content in the detection blood according to claim 1, it is characterized in that: the mol ratio of said nucleic acid molecule and protoheme and luminol reagent is 1: 10: 1000.

3. the reagent of potassium content in the detection blood according to claim 1, it is characterized in that: said nucleic acid molecule is following:

Sequence 5′-GGG-ATT-GGG-ATT-GGG-ATT-GGG 5′-GGGG-ATT-GGGG-ATT-GGGG-ATT-GGGG 5′-GGGGG-ATT-GGGGG-ATT-GGGGG-ATT-GGGGG 5′-TAATGGGG-ATT-GGGG-ATT-GGGG-ATT-GGGG-TAAT 5′-ACTTAATGGGG-ATT-GGGG-ATT-GGGG-ATT-GGGGACTTAAT

4. the preparation method of the reagent of potassium content in the detection blood as claimed in claim 1 is characterized in that: comprise the steps:

(1) Z 150PH-water-sol of phenyl vinylpyridine, nucleic acid molecule, protoheme, luminol reagent are mixed the back and become collosol state;

(2) with the mixture of step (1) by uv irradiating, the phenyl vinylpyridine produces crosslinked, makes mixture solidified, nucleic acid molecule, protoheme, luminol reagent in gel, are formed detection layers by physical fixation;

The mol ratio of the water-sol of said Z 150PH-phenyl vinylpyridine, nucleic acid molecule, protoheme, luminol reagent is 1: 10: 1000.

5. the preparation method of the reagent of potassium content in the detection blood according to claim 4; It is characterized in that: the lower floor in detection layers is shaped on the hydrogel supporting layer in addition; Composition is the linear polymer of polyoxyethylene glycol and polyesteramide, is shaped on the carbon black resist on the upper strata of detection layers.

6. the preparation method of the reagent of potassium content in the detection blood according to claim 5, it is characterized in that: concrete preparation process is following:

(1) dissolving nucleic acid molecule and protoheme in the HEPES of 50mM buffered soln pH 7.0 are heated to 80 ℃ with the solution with water bath and also are cooled to room temperature at normal temperatures so that the molecular structure homogenization of nucleic acid adds luminol reagent in solution;

(2) on plastics film, be coated with the film of last layer D4 hydrogel and make it natural air drying with the scraper film applicator, thickness is 90-110 μ m;

(3) in 10wt%PVA-Sbq solution, add the solution of step (1) preparation and stirring,

(4) on the film of the D4 hydrogel of step (2), be coated with last layer step (3) and obtain solution, uv lamp is placed top 25-35 minute of rete, make phenylpyridine ethene crosslinked and rete is become dry;

(5) the last the superiors are coated with last layer with the scraper film applicator and contain 10wt% sooty D6 hydrogel and make the rete natural air drying.

7. one kind contains the test kit that detects the reagent of potassium content in the blood according to claim 1.

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