Quantitative detection method of rare earth doped calcium phosphate fluorescent nanoparticles in organisms

Abstract

A quantitative detection method of rare earth doped calcium phosphate fluorescent nanoparticles (RE-nCaP) in organisms includes establishing a fluorescent intensity-concentration standard curve of rare earth ions, preparing samples to be tested and the blank control group into homogenate, performing centrifuging and testing the fluorescent intensity of supernatants, calculating the fluorescent intensity values per unit mass or volume of the samples and the blank control group, and performing significant difference analysis; if P is greater than or equal to 0.05, determining that the RE-nCaP content in the samples is 0, and if P is smaller than 0.05, testing the tissue extraction rate of RE-nCaP; and comprehensively considering the tissue extraction rate, the homogenate volume, the fluorescent intensity value per until mass or volume, the homogenate dilution ratio, and the doping amount to obtain the accurate content of the RE-nCaP in biological tissue samples.

Claims

1. A quantitative detection method of rare earth doped calcium phosphate fluorescent nanoparticles (RE-nCaP) in at least one biological tissue of one of blood, heart, liver, spleen, lung, kidney, pancreas, brain, lymph, skin, and nerve of an animal, comprising steps of: (a) establishing a fluorescent intensity-concentration standard curve of rare earth (RE) ions in a fluorescent enhancement liquid, wherein the fluorescent intensity-concentration standard curve satisfies a relationship of Y=B+N.Math.X, wherein Y and X are respectively a fluorescent intensity and an RE concentration of the fluorescent enhancement liquid, and B and N are constants and respectively an intercept and a slope of the fluorescent intensity-concentration standard curve; (b) separately treating at least one test biological tissue sample of a group to be tested, and at least one control biological tissue sample of a blank control group with an acid solution to obtain homogenates of the group to be tested and the blank control group, wherein the at least one test biological tissue sample of the group to be tested is obtained from said at least one biological tissue of the animal, and the at least one control biological tissue sample of the blank control group is obtained from at least one control biological tissue of another animal containing no RE-nCaP, wherein said at least one control biological tissue is a tissue of one of blood, heart, liver, spleen, lung, kidney, pancreas, brain, lymph, skin, and nerve of said another animal that is corresponding to said one of said blood, heart, liver, spleen, lung, kidney, pancreas, brain, lymph, skin, and nerve of the animal; performing centrifugal separation on the homogenates of the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group separately to obtain supernatants of the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group; diluting the supernatants of the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group with the fluorescent enhancement liquid; detecting fluorescent intensities of the diluted supernatants of the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group; calculating values, T and T.sub.0, of a fluorescent intensity per unit mass or volume of the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group according to formula (I): $\begin{array}{cc}T=\frac{y-B}{W};\mathrm{and}{T}_0=\frac{y_0-B}{W_0}& \left(I\right)\end{array}$ wherein y and y.sub.0 are respectively the detected fluorescent intensities of the diluted supernatants of the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group, W is a weight or volume of the at least one test biological tissue sample of the group to be tested, and W.sub.0 is a weight or volume of the at least one control biological tissue sample of the blank control group; performing significant difference analysis on the values T and T.sub.0 of the fluorescent intensity per unit mass or volume between the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group, so as to determine if the at least one test biological tissue sample in the group to be tested contain the RE-nCaP; and continuing to perform a step (c) and a step (d) to further determine an amount of the RE-nCaP when the at least one test biological tissue sample in the group to be tested are determined to contain the RE-nCaP; (c) separately adding the RE-nCaP to the homogenate of the at least one control biological tissue sample of the blank control group and to the acid solution having a same volume as that of the homogenate of the at least one control biological tissue sample of the blank control group; performing centrifugal separation on the RE-nCaP added homogenate of the at least one control biological tissue sample of the blank control group and the RE-nCaP added acid solution separately to obtain supernatants of the RE-nCaP added homogenate of the at least one control biological tissue sample of the blank control group and the RE-nCaP added acid solution; diluting the supernatants of the RE-nCaP added homogenate of the at least one control biological tissue sample of the blank control group and the RE-nCaP added acid solution with the fluorescent enhancement liquid; detecting fluorescent intensities of the diluted supernatants of the RE-nCaP added homogenate of the at least one control biological tissue sample of the blank control group and the RE-nCaP added acid solution; calculating a tissue retention rate, S, per unit mass or volume according to formula (II): $\begin{array}{cc}S=\frac{\left(A_0-A_1\right)}{A_0{W}_0}& \left(\mathrm{II}\right)\end{array}$ wherein A.sub.1 is the detected fluorescent intensity of the RE-nCaP added homogenate of the at least one control biological tissue sample of the blank control group and A.sub.0 is the detected fluorescent intensity of the RE-nCaP added acid solution, and W.sub.0 is the weight or volume of the at least one control biological tissue sample of the blank control group; and calculating a tissue extraction rate, R, of the group to be tested according to a weight or volume of the at least one test biological tissue sample in the group to be tested according to formula (III):
R=(1S.Math.W)100%(III) wherein W is the weight or volume of the at least one test biological tissue sample in the group to be tested; and (d) calculating an amount, M, of the RE-nCaP in the at least one test biological tissue sample of the group to be tested according to formula (IV): $\begin{array}{cc}M=\left(T_1-\stackrel{\_}{T_0}\right).Math.\frac{kV}{\mathrm{RN}}& \left(\mathrm{IV}\right)\end{array}$ wherein T.sub.1 is the value of the fluorescent intensity per unit mass or volume of the at least one test biological tissue sample of the group to be tested, T.sub.0 is an average value of the fluorescent intensity per unit mass or volume of the at least one control biological tissue sample of the blank control group, k is a ratio of a dilution ratio of the homogenate of the at least one test biological tissue sample to the diluted supernatants of the at least one test biological tissue sample to a mole amount of rare earth elements in calcium phosphate, V is a homogenate volume of the at least one test biological tissue sample in the group to be tested, and R is the tissue extraction rate R of the group to be tested.

2. The method according to claim 1, wherein the step (a) of establishing the fluorescent intensity-concentration standard curve comprises: by using the fluorescent enhancement liquid as a solvent, preparing a series of rare earth ion standard solutions of different concentrations; determining fluorescent intensities of the rare earth ion standard solutions at a specific emission wavelength under a specific excitation wavelength by a fluorescent spectrophotometer; and performing fitting of the determined fluorescent intensities and the different concentrations of the rare earth ion standard solutions so as to obtain the fluorescent intensity-concentration standard curve.

3. The method according to claim 1, wherein in the step (b), said separately treating the at least one test biological tissue sample of the group to be tested, and the at least one control biological tissue sample of the blank control group with the acid solution comprises: measuring a mass or volume of the at least one test biological tissue sample in the group to be tested and a mass or volume of the at least one control biological tissue sample in the blank control group separately; adding the acid solution to the at least one test biological tissue sample in the group to be tested and the at least one control biological tissue sample in the blank control group separately to obtain the homogenates of the group to be tested and the blank control group; taking an amount of the homogenate in the group to be tested and an amount of the homogenate in the blank control group, wherein the amount of the homogenate in the group to be tested and the amount of the homogenate in the blank control group are equivalent; said diluting the supernatants of the group to be tested and the blank control group with the fluorescent enhancement liquid comprises: mixing the supernatants of the group to be tested and the blank control group with the acid solution according to a proportion to obtain supernatant diluents of the group to be tested and the blank control group, respectively; mixing the supernatant diluents of the group to be tested and the blank control group with the fluorescent enhancement liquid according to a proportion to obtain mixed liquids of the group to be tested and the blank control group, respectively; and said performing the significant difference analysis on the values T and T.sub.0 of the fluorescent intensities per unit mass or volume between the at least one test biological tissue sample of the group to be tested and the at least one control biological tissue sample of the blank control group comprises: when P is greater than or equal to 0.05, indicating that significant difference does not exist between the at least one test biological tissue sample in the group to be tested and the at least one control biological tissue sample in the blank control group, and determining that the at least one test biological tissue sample in the group to be tested does not contain the RE-nCaP; and when P is smaller than 0.05, indicating that the significant difference exists between the at least one test biological tissue sample in the group to be tested and the at least one control biological tissue sample in the blank control group, determining that the at least one test biological tissue sample in the group to be tested contains the RE-nCaP.

4. The method according to claim 1, wherein the rare earth ions are Eu.sup.3+ or Tb.sup.3+, and a rare earth ion doping manner is separate doping or codoping with other rare-earth elements.

5. The method according to claim 1, wherein a value range of a molar ratio of RE/(RE+Ca) in the RE-nCaP is 0.1%-18%.

6. The method according to claim 1, wherein the acid solution is a nitric acid aqueous solution or a hydrochloric acid aqueous solution, and a concentration of the acid solution is 0.5-5 mol/L.

7. The method according to claim 1, wherein a fluorescent intensity of a sample solution at a specific emission wavelength under a specific excitation wavelength is detected through a fluorescent spectrophotometer, the specific excitation wavelength is 330-350 nm, and the specific emission wavelength is 618 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the fluorescent intensity-concentration standard curve of Eu ions established in the quantitative detection method disclosed by the invention.

(2) FIG. 2 is the detection result of the fluorescent intensity value of the livers per unit mass in the group to be tested and the blank control group in the embodiment 1.

(3) FIG. 3 is the detection result of the fluorescent intensity value of the kidneys per unit mass in the group to be tested and the blank control group in the embodiment 2.

(4) FIG. 4 is the detection result of the fluorescent intensity value of the pancreases per unit mass in the group to be tested and the blank control group in the embodiment 3.

(5) FIG. 5 is the detection result of the fluorescent intensity value of the blood per unit volume in the group to be tested and the blank control group in the embodiment 4.

(6) FIG. 6 is the detection result of the fluorescent intensity value of the hearts per unit weight in the group to be tested and the blank control group in the embodiment 5.

(7) FIG. 7 is the detection result of the fluorescent intensity value of the tumors per unit mass in the group to be tested and the blank control group in the embodiment 6.

DETAILED DESCRIPTION OF THE INVENTION

(8) In order to enable those skilled in the art to sufficiently understand the technical scheme and the beneficial effects of the quantitative detection method disclosed by the invention, further description is performed through combination with concrete embodiments.

(9) The molar ratio of europium ion doped hydroxylapatite nanoparticles (Eu-nCaP, Eu/(Eu+Ca) used in the quantitative detection method disclosed by the invention is 4%, and the chemical molecular formula is Eu.sub.0.4Ca.sub.9.6(PO.sub.4).sub.6(OH).sub.2). The europium ion doped hydroxylapatite nanoparticles are prepared according to a method disclosed by the Chinese patent CN105018086A (rare earth doped calcium phosphate fluorescent nanoparticles and preparation method thereof, Hanyingchao, et al), the Chinese patent CN107573938A (preparation method and application of rare earth doped apatite fluorescent nanometer points, Hanyingchao, et al). Other rare earth ion (such as Tb) doped calcium phosphate nanoparticles can also be prepared by the method.

(10) The biological tissue samples (livers, kidneys, pancreases, hearts, blood, tumors and the like) involved by the invention all come from nude mice, and all nude mouse experiments are all strictly executed according to Regulations for the Administrations of Affairs Concerning Experimental Animals in China and the regulations of relevant international laws and regulations. In embodiments 1-5, normal nude mice of which the weight is about 20 g are used for experiments, in embodiment 6, nude mice (of which the weight is about 20 g) having a subcutaneous transplanted tumor model (HepG2 cells) are used for experiments, and the size of the tumors is 200-300 mm.sup.3. In each embodiment, 6 nude mice in the same batch are used, and are randomly divided into 2 groups which are recorded as the group to be tested (an experimental group) and the blank control group. The fluorescent enhancement liquid used in the quantitative detection method disclosed by the invention is the DELFIA Enhancement Solution, and the manufacturer is PerkinElmer Inc.

(11) The method for establishing the fluorescent intensity-concentration standard curve of rare earth ions disclosed by the invention specifically comprises the following steps of precisely weighing 36.641 mg of europium chloride hexahydrate, and dissolving the weighed europium chloride hexahydrate in 100 mL of ultra pure water to obtain europium ion storing liquid of which the concentration is 1 mmol/L; diluting the europium ion storing liquid into europium ion solutions of different concentrations (0.50000, 0.99502, 1.48522, 1.97044, 2.45098, 2.92654 and 3.86473, nmol/L) with the fluorescent enhancement liquid, and then determining the fluorescent intensity of the europium ion solutions of different concentrations at 618 nm under the condition that the excitation wavelength is 340 nm with the fluorescent spectrophotometer; and drawing the relationship curve of the fluorescent intensity y and the concentration x in a rectangular coordinate system, and performing linear fitting so as to obtain y=B+Nx (R.sup.2=0.9960), wherein B is intercept (cps), the value of the intercept is 0.039, N is slope (cps/(nmol/L)), the value of the slope is 1.501, and the result is as shown in the FIG. 1. The standard curve is uniformly used in all embodiments in the invention.

Embodiment 1

(12) An Eu-nCaP aqueous suspension (200 L, 5 mg/mL) is injected into the nude mice in the group to be tested through caudal vein injection, and timing immediately starts. The nude mice in the blank control group are not subjected to injection treatment. 8 h later, the liver tissues of the nude mice in the group to be tested and the liver tissues of the nude mice in the blank control group are taken out and are thoroughly rinsed with a PBS solution, and 3 liver samples are obtained in each group (namely that 3 liver samples are obtained in the group to be tested, and 3 liver samples are obtained in the blank control group). After surface moisture is thoroughly absorbed with paper, the weight W(g) of each liver sample is weighed, wherein W.sub.1 in the group to be tested is sequentially 0.097 g, 0.070 g and 0.074 g, and W.sub.0 in the blank control group is sequentially 0.054 g, 0.102 g and 0.085 g. The tissue samples of the group to be tested and the blank control group are prepared into 1 mL of liver homogenate with 2 mol/L nitric acid. 0.5 mL of the liver homogenate in the group to be tested and 0.5 mL of the liver homogenate in the blank control group are separately taken, and are put in centrifugal tubes, high speed centrifugation (5000 r/min) is performed, 40 L of supernatant and 760 L of nitric acid (2 mol/L) are mixed so that a supernatant diluent is prepared. 5 L of the supernatant diluent and 995 L of fluorescent enhancement liquid are mixed so that mixed liquid is prepared, and the fluorescent intensity of the mixed liquid at the emission wavelength of 618 nm (Ex=340 nm) is tested. The fluorescent intensity value y.sub.1 in the group to be tested is sequentially 10.068 cps, 11.244 cps and 10.463 cps, and the fluorescent intensity value y.sub.0 in the blank control group is sequentially 0.780 cps, 0.080 cps and 0.653 cps. The fluorescent intensity value (T) of the livers per unit mass in the group to be tested and the blank control group is calculated according to the formula (I):

(13) $\begin{array}{cc}T=\frac{y-B}{W},& \left(I\right)\end{array}$
The fluorescent intensity value T.sub.1 of the livers per unit mass in the group to be tested is sequentially 103.392 cps/g, 160.071 cps/g and 140.865 cps/g, and the fluorescent intensity value T.sub.0 of the livers per unit mass in the blank control group is sequentially 13.722 cps/g, 0.402 cps/g and 7.224 cps/g; through single factor variance analysis, compared with the T.sub.0 value in the blank control group, the T.sub.1 value in the group to be tested has significance difference (P=0.0017<0.05), and the result is seen in the FIG. 2. It can be seen that the content of Eu-nCaP in the biological tissue samples in the group to be tested is determined through subsequent steps.

(14) The extraction rate of the Eu-nCaP in the livers is calculated through the following steps: separately putting the homogenate in the blank control group and nitric acid (2 mol/L) having the same volume (500 L) as that of the homogenate in the blank control group in the centrifugal tubes, separately adding 760 L of Eu-nCaP suspension liquid (0.25 g/mL), performing uniform mixing, allowing the mixtures to stand for 4 h, and performing centrifuging; taking 40 L, of supernatants and 760 L of nitric acid (2 mol/L), and performing mixing to obtain supernatant diluents; taking 5 L of the supernatant diluent and 995 L of the fluorescent enhancement liquid, and performing mixing so as to obtain test liquid; testing the fluorescent intensity of the test liquid at the emission wave length of at 618 nm (Ex=340 nm), wherein the fluorescent intensity A.sub.1 of the homogenate in the blank control group is 2.375 cps, and the fluorescent intensity A.sub.0 in a nitric acid group is 2.421 cps, and calculating the tissue retention rate S per unit mass according to the formula (II):

(15) $\begin{array}{cc}S=\frac{\left(A_0-A_1\right)}{A_0{W}_0},& \left(\mathrm{II}\right)\end{array}$
Based on the calculated S=0.085, further calculating the tissue extraction rate R according to the formula (III):
R=(1S.Math.W.sub.1)100%,(III)
so that the liver extraction rate R in 3 groups to be tested is 97.8%, 98.4% and 98.3% sequentially. The content of the Eu-nCaP in the livers in the group to be tested is calculated through the following steps of according to the known fluorescent intensity-concentration standard curve, the fluorescent intensity value (T.sub.1) of the livers per unit mass in the group to be tested, the average value (T.sub.0) of the fluorescent intensity value of the livers per unit mass in the blank control group, the dilution ratio of the sample homogenate to the test liquid, the liver extraction rate and the chemical molecular formula of europium doped hydroxylapatite, and calculating the content M of Eu-nCaP in the liver tissue of the group to be tested according to the formula (IV):

(16) $\begin{array}{cc}M=\left(T_1-\stackrel{\_}{T_0}\right).Math.\frac{kV}{\mathrm{RN}},& \left(\mathrm{IV}\right)\end{array}$
wherein in the embodiment, k is 4000/0.4=10.sup.4, V=1*10.sup.3 L, and T.sub.0=7.116; the content of the Eu-nCaP in the liver tissue of the group to be tested is calculated to be 655.627 nmol/g, 1035.222 nmol/g and 906.053 nmol/g respectively; and therefore, the content of the Eu-nCaP in the liver tissue in the group to be tested is 865.634192.999 nmol/g.

Embodiment 2

(17) The kidneys of the nude mice in the group to be tested and the kidneys of the nude mice in the control group are obtained by the method in the embodiment 1, and 3 samples are contained in each group (3 samples are contained in the group to be tested, and 3 samples are contained in the blank control group); the weight W(g) of each kidney sample is weighed through a scales, wherein W.sub.1 in the group to be tested is sequentially 0.073 g, 0.054 g and 0.065 g, and W.sub.0 in the blank control group is sequentially 0.020 g, 0.161 g and 0.097 g; and the tissue samples of the group to be tested and the blank control group are prepared into 1 mL of kidney homogenate through the 2 mol/L nitric acid.

(18) By the method in the embodiment 1, the kidney homogenate in the group to be tested and the kidney homogenate in the blank control group are prepared into mixed liquid, the fluorescent intensity value y of the mixed liquid is tested, the fluorescent intensity value y.sub.1 in the group to be tested is sequentially 4.555 cps, 2.822 cps and 2.498 cps, and the fluorescent intensity value y.sub.0 in the blank control group is sequentially 0.282 cps, 0.174 cps and 0.095 cps; through the formula (I), the T.sub.1 value in the group to be tested is calculated out to be 61.863 cps/g, 51.537 cps/g and 37.831 cps/g sequentially, and the T.sub.0 value in the blank control group is calculated out to be 1.215 cps/g, 0.839 cps/g and 0.577 cps/g sequentially; through single factor variance analysis, compared with the T.sub.0 value in the blank control group, the T.sub.1 value in the group to be tested has significant difference (P=0.0021<0.05), and the result is seen in the FIG. 3; and it can be seen that the content of the Eu-nCaP in the group to be tested is further calculated through subsequent steps.

(19) According to the method in the embodiment 1, the fluorescent intensity value of the homogenate in the blank control group and the fluorescent intensity value of the nitric acid are tested, the retention rate S is calculated according to the formula (II), and further the extraction rate R of the Eu-nCaP in the kidneys is calculated according to the formula (III), wherein A.sub.1=2.279 cps, A.sub.0=2.421 cps and W.sub.0=0.097 g; and therefore, the kidney extraction rate R in the 3 groups to be tested is calculated to be 95.6%, 96.7% and 96.1%.

(20) The content of the Eu-nCaP in the kidneys in the group to be tested is calculated through the following step: according to the formula (IV) (in the embodiment, B=0.039, N=1.501, V=1*10.sup.3L, k=10.sup.4, T.sub.0=0.877), calculating the content of the Eu-nCaP in the kidneys in the group to be tested to be 425.065, 348.901 and 256.267 nmol/g respectively, thereby obtaining the content of the Eu-nCaP in the kidney tissue of the group to be tested being 343.41184.533 nmol/g.

Embodiment 3

(21) The pancreases of the nude mice in the group to be tested and the pancreases of the nude mice in the control group are obtained by the method in the embodiment 1, and 3 samples are contained in each group (3 samples are contained in the group to be tested, and 3 samples are contained in the blank control group); the weight W(g) of each pancrease sample is weighed through a scales, wherein W.sub.1 in the group to be tested is sequentially 0.027 g, 0.029 g and 0.028 g, and W.sub.0 in the blank control group is sequentially 0.050 g, 0.033 g and 0.034 g; and the tissue samples in the group to be tested and the blank control group are prepared into 1 mL of pancrease homogenate through the 2 mol/L nitric acid.

(22) According to the method in the embodiment 1, the pancrease homogenate the group to be tested and the pancrease homogenate in the blank control group are prepared into mixed liquid, the fluorescent intensity value of the mixed liquid is tested, the fluorescent intensity value y.sub.1 in the group to be tested is sequentially 1.245 cps, 1.256 cps and 1.320 cps, and the fluorescent intensity value y.sub.0 in the blank control group is sequentially 0.312 cps, 0.491 cps and 0.495 cps; through the formula (I), the T.sub.1 value in the group to be tested is calculated to be 44.667, 41.966, 45.750 cps/g sequentially, and the T.sub.0 value in the blank control group is calculated to be 5.460, 13.679, 13.412 cps/g sequentially; through single factor variance analysis, compared with the T.sub.0 value in the blank control group, the T.sub.1 value in the group to be tested has significant difference (P=0.0003<0.05), and the result is seen in the FIG. 4; and it can be seen that the content of the Eu-nCaP in the group to be tested is further determined through subsequent steps.

(23) According to the method in the embodiment 1, the fluorescent intensity value of the homogenate in the blank control group and the fluorescent intensity value of the nitric acid are tested, the retention rate S is calculated according to the fluorescent intensity value, and the extraction rate R of the Eu-nCaP in the pancreases is further calculated, wherein A.sub.1=2.032 cps, A.sub.0=2.421cps and W.sub.0=0.050 g; and therefore, the pancrease extraction rate R in the 3 groups to be tested is calculated to be 91.32%, 90.68% and 91.00% from the formula (III).

(24) The content of the Eu-nCaP in the pancreases in the group to be tested is calculated through the following step: according to the formula (IV) (in the embodiment, B=0.039, N=1.501, V=1*10.sup.3, k=10.sup.4, T.sub.03210.856), calculating the content of the Eu-nCaP in the kidneys in the group to be tested to be 246.656 nmol/g, 228.559 nmol/g and 255.458 nmol/g respectively, thereby obtaining the content of the Eu-nCaP in the pancrease tissue of the group to be tested being 243.55713.715 nmol/g.

Embodiment 4

(25) Reference to the method in the embodiment 1, 0.04 mL (W) of blood of the nude mice in the group to be tested and 0.04 mL (W) of blood of the nude mice in the control group are separately collected, and the blood of the nude mice in the group to be tested and 1 mL of nitric acid (2 mol/L) are mixed and uniformly shaken, the blood of the nude mice in the control group and 1 mL of nitric acid (2 mol/L) are mixed and uniformly shaken, so that blood homogenates are prepared. 3 samples are contained in each group (3 samples are contained in the group to be tested, and 3 samples are contained in the blank control group).

(26) According to the method in the embodiment 1, the fluorescent intensity of the blood homogenate in the group to be tested and the blood homogenate in the blank control group are tested, the fluorescent intensity y.sub.1 in the group to be tested is sequentially 2.642 cps, 2.720 cps and 2.810 cps, and the fluorescent intensity value y.sub.0 in the blank control group is sequentially 0.210 cps, 0.241 cps and 0.196 cps; through the formula (I), the T.sub.1 value in the group to be tested is calculated to be 65.075, 67.025, 69.275 cps/mL sequentially, and the T.sub.0 value in the blank control group is calculated to be 4.275, 5.050, 3.925 cps/mL sequentially; through single factor variance analysis, compared with the T.sub.0 value in the blank control group, the T.sub.1 value in the group to be tested has significant difference (P=0.000001<0.05), and the result is seen in the FIG. 5; and it can be seen that the content of the Eu-nCaP in the group to be tested is further determined through subsequent steps.

(27) According to the method in the embodiment 1, the fluorescent intensity value of the homogenate in the blank control group and the fluorescent intensity value of the nitric acid is tested, the retention rate S is calculated according to the fluorescent intensity value, and the extraction rate R of the Eu-nCaP in the blood is further calculated, wherein A.sub.1=2.323 cps, A.sub.0=2.421 cps and W.sub.0=0.04 mL; and therefore, the blood extraction rate R in the 3 groups to be tested is calculated from the formula (III) to be 98.9%.

(28) The content of the Eu-nCaP in the blood in the group to be tested is calculated through the following step: according to the formula (IV) (in the embodiment, B=0.039, N=1.501, V=1*10.sup.3, k=10.sup.4, T.sub.0=4.417), calculating the content of the Eu-nCaP in the blood in the group to be tested to be 409.026 nmol/mL, 422.175 nmol/mL and 437.347 nmol/mL respectively, thereby obtaining the content of the Eu-nCaP in the blood tissue of the group to be tested being 422.84914.173 nmol/mL.

Embodiment 5

(29) The hearts of the nude mice in the group to be tested and the hearts of the nude mice in the control group are obtained by the method in the embodiment 1, and 3 samples are contained in each group (3 samples are contained in the group to be tested, and 3 samples are contained in the blank control group); the weight W(g) of each heart sample is weighed through a scales, wherein W.sub.1 in the group to be tested is sequentially 0.027 g, 0.024 g and 0.012 g, and W.sub.0 in the blank control group is sequentially 0.018 g, 0.063 g and 0.020 g; and the tissue samples in the group to be tested and the blank control group are prepared into 1 mL of heart homogenate through the 2 mol/L nitric acid.

(30) According to the method in the embodiment 1, the heart homogenate in the group to be tested and the heart homogenate in the blank control group are prepared into mixed liquid, the fluorescent intensity value of the mixed liquid is tested, the fluorescent intensity value y.sub.1 in the group to be tested is sequentially 0.133 cps, 0.410 cps and 0.120 cps, and the fluorescent intensity value y.sub.0 in the blank control group is sequentially 0.475cps, 0.117cps and 0.487cps; through the formula (I), the T.sub.1 value in the group to be tested is calculated to be 3.443, 15.394, 6.750 cps/g sequentially, and the T.sub.0 value in the blank control group is calculated to be 24.222, 1.238, 22.400 cps/g sequentially; through single factor variance analysis, P=0.416>0.05, indicating that compared with the T.sub.0 value in the blank control group, the T.sub.1 value in the group to be tested has no significant difference, and the result is seen in the FIG. 6; and therefore, the content of the Eu-nCaP in the heart tissue is determined to be 0 nmol/g.

Embodiment 6

(31) The tumor tissue of the nude mice in the group to be tested and the tumor tissue of the nude mice in the control group are obtained by the method in the embodiment 1, and 3 samples are contained in each group (3 samples are contained in the group to be tested, and 3 samples are contained in the blank control group); the weight W(g) of each tumor sample is weighed through a scales, wherein W.sub.1 in the group to be tested is sequentially 0.037 g, 0.027 g and 0.026 g, and W.sub.0 in the blank control group is sequentially 0.037 g, 0.115 g and 0.022 g; and the tissue samples in the group to be tested and the blank control group are prepared into 1 mL of tumor homogenate through the 2 mol/L nitric acid.

(32) According to the method in the embodiment 1, the tumor homogenate in the group to be tested and the tumor homogenate in the blank control group are prepared into mixed liquid, the fluorescent intensity value of the mixed liquid is tested, the fluorescent intensity value y.sub.1 in the group to be tested is sequentially 0.848, 0.810 and 0.861 cps, and the fluorescent intensity value y.sub.0 in the blank control group is sequentially 0.368, 0.516 and 0.210 cps; through the formula (I), the T.sub.1 value in the group to be tested is calculated to be 21.865 cps/g, 28.556 cps/g and 31.615 cps/g sequentially, and the T.sub.0 value in the blank control group is calculated to be 8.892 cps/g, 4.148 cps/g and 7.773cps/g sequentially; through single factor variance analysis, compared with the T.sub.0 value in the blank control group, the T.sub.1 value in the group to be tested has significant difference (P=0.0032<0.01), and the result is seen in the FIG. 7; and it can be seen that the content of the Eu-nCaP in the group to be tested is further determined through subsequent steps.

(33) According to the method in the embodiment 1, the fluorescent intensity value of the homogenate in the blank control group and the fluorescent intensity value of the nitric acid are tested, the retention rate S is calculated according to the fluorescent intensity value, and further the extraction rate R of the Eu-nCaP in the tumors is calculated, wherein A.sub.1=2.323 cps, A.sub.0=2.421 cps and W.sub.0=0.115 g; and therefore, the tumor extraction rate R in the 3 groups to be tested is calculated from the formula (III) to be 98.70%, 99.05% and 99.08%.

(34) The content of the Eu-nCaP in the tumors in the group to be tested is calculated through the following step: according to the formula (IV) (in the embodiment, B=0.039, N=1.501, V=1*10.sup.3, k=10.sup.4, T.sub.0=6.937), calculating the content of the Eu-nCaP in the group to be tested to be 100.765 nmol/g, 145.410 nmol/g and 165.932 nmol/g respectively, thereby obtaining the content of the Eu-nCaP in the tumor tissue of the group to be tested being 137.36933.319 nmol/g.