BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
检测原理：无需处理样本，利用无机磷与钼酸铵结合生成磷钼酸铵，通过紫外分光光度计检测325～340nm处吸光度，根据公式计算出无机磷含量。紫外法优点在于：操作简单、快速、稳定；缺点在于：易受溶血、黄疸、脂血的干扰。该试剂盒仅用于科研领域，不宜用于临床诊断或其他用途。
| I1510435 | Component | 100T | Storage |
| I1510435A | 磷标准 (1mg/mL) | 1 mL | 2-8℃. |
| I1510435B | 磷标准稀释液 | 2 mL | RT. |
| I1510435C | 钼酸铵粉末 | 1 EA | RT. |
| I1510435D | 钼酸铵稀释液 (2×) | 100 mL | RT. |
自备仪器和试剂
操作步骤（仅供参考）
1、（选做）制备样品
1.1 血浆、血清样品
血浆、血清按照常规方法制备，可以直接用于本试剂盒的测定，-20℃冻存，用于Pi的检测。
1.2 细胞或组织样品
取恰当细胞或组织进行匀浆，低速离心取上清，-20℃冻存，用于Pi的检测。
1.3 高浓度样品
如果样品中含有较高浓度的Pi，可以使用ddH₂O稀释，不宜使用普通蒸馏水稀释。
注：（选做）样品准备完毕后可以用BCA蛋白浓度测定试剂盒测定蛋白浓度，以便于后续计算单位蛋白重量组织或细胞内的Pi含量。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
2、制备磷标准工作液
取适量的磷标准 (1mg/mL)，按磷标准 (1mg/mL)：磷标准稀释液=1：39的比例稀释，即获得磷标准工作液 (25 μg/mL=0.807 mmol/L)，4℃保存，用于Pi的检测。
3、制备钼酸铵显色液
称取适量的钼酸铵粉末，按照钼酸铵粉末：钼酸铵稀释液 (2×)：去离子水=12mg：10ml：10ml的比例混合溶解，即可使用。亦可称取120mg钼酸铵粉末加入100ml去离子水，充分溶解后分装成10ml或适当规格的小份，使用时与钼酸铵稀释液 (2×) 等比例混合即可使用，-20℃保存，可适当延长保质期。钼酸铵显色液配制后可4℃保存2周，如溶液变浑浊或颜色加深则应弃用。请注意，本产品提供多于实际用量的钼酸铵粉末。
4、Pi加样
按照下表设置空白管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡，小心混匀。如果样品中的无机磷浓度过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置2平行管，求平均值。
加入物（mL） | 空白管 | 标准管 | 测定管 |
ddH₂O | 0.05 | — | — |
磷标准工作液 (25 μg/mL) | — | 0.05 | — |
待测样品 | — | — | 0.05 |
钼酸铵显色液 | 2 | 2 | 2 |
5、Pi测定：混匀，室温静置5min，比色杯光径1cm，分光光度计340nm处检测，以空白管调零，读取标准管、测定管的吸光度 （即为A<sub>标准</sub>，A<sub>测定</sub>）。
6、计算
6.1 血清、血浆中无机磷计算公式：磷=(A测定/A标准)×0.807×N(mmol/L)=(A测定/A标准)×25×N (mg/L)
6.2 尿液中无机磷计算公式：磷=(A测定/A标准)×0.807×N(mmol/L)=(A测定/A标准)×25×N (mg/L)
6.3 组织中磷计算公式：磷 (mmol/mg)=(A测定/A标准)×0.807×N/c
参数说明：
A测定：测定管的吸光度
A标准：标准管的吸光度
N：样品稀释倍数
c：待测样品蛋白浓度 (mg/L)
单位换算：1 mg/dL=10 mg/L=1 mmol/L×0.323
参考区间
健康成年人血清磷浓度：0.9～1.34 mmol/L (2.76～4.16 mg/dl)
注意事项
1、本法应在5min～2h内检测，3h后标准管吸光度无变化，测定管吸光度随着时间的延长而上升。
2、溶血样本对检测有干扰，尽量避免采用溶血样本。
3、黄疸和脂血样本应做标本空白。
4、本法能够用于自动生化分析仪终点检测法。
5、钼酸铵显色液配制后可4℃保存2周，如溶液变浑浊或颜色加深则应弃用。
6、如果样品浓度过高，应用蒸馏水稀释后重测，结果乘以稀释倍数。
7、340nm测得的吸光度是325nm的82%。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Inorganic Phosphorus in Serum primarily consists of two phosphate anions, H₂PO₄⁻ and HPO₄²⁻, which can rapidly interconvert depending on the pH environment. In serum at pH 7.4, the concentration ratio of these two anions is approximately 1:4. Under acidosis, the ratio is about 1:1, while under alkalosis, it shifts to 1:9. In urine at pH 4.5, the concentration ratio is about 100:1. The conventional detection method recommended by the WHO is colorimetry. The National Center for Clinical Laboratories of China recommends the ferrous sulfate molybdenum blue method and the metol molybdenum blue method as routine methods. UV spectrophotometry can also be employed.
Detection Principle
Without the need for sample pretreatment, inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate. The absorbance is directly measured at 325–340 nm using a UV spectrophotometer, and the inorganic phosphorus content is calculated according to the formula. Advantages of the UV method include simplicity, speed, and stability. Its disadvantages include susceptibility to interference from hemolysis, icterus, and lipemia. This kit is intended for research purposes only and is not suitable for clinical diagnosis or other applications.
| I1510435 | Component | 100T | Storage |
| I1510435A | Phosphorus Standard (1 mg/mL) | 1 mL | 2-8℃. |
| I1510435B | Phosphorus Standard Diluent | 2 mL | RT. |
| I1510435C | Ammonium Molybdate Powder | 1 EA | RT. |
| I1510435D | Ammonium Molybdate Diluent (2×) | 100 mL | RT. |
Reagents, consumables and Equipments not provided
Procedure (For Reference Only)
1. (Optional) Sample Preparation
1.1 Plasma/Serum Samples
Plasma or serum samples prepared by conventional methods can be used directly for assay with this kit. Store at -20°C for Pi detection.
1.2 Cell or Tissue Samples
Homogenize an appropriate amount of cells or tissue. After low-speed centrifugation, collect the supernatant and store at -20°C for Pi detection.
1.3 High-Concentration Samples
If the sample contains a high concentration of Pi, dilute it with ddH₂O. Regular distilled water is not recommended for dilution.
Note: (Optional) After sample preparation, the protein concentration can be measured using a BCA Protein Assay Kit to facilitate subsequent calculation of Pi content per unit protein weight in tissues or cells. The Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Assay Kit is recommended.
2. Preparation of Phosphorus Standard Working Solution
Take an appropriate amount of the Phosphorus Standard (1 mg/mL) and dilute it with the Phosphorus Standard Diluent at a ratio of 1:39 (Phosphorus Standard: Diluent) to obtain the Phosphorus Standard Working Solution (25 μg/mL = 0.807 mmol/L). Store at 4°C for Pi detection.
3. Preparation of Ammonium Molybdate Chromogenic Solution
Weigh an appropriate amount of Ammonium Molybdate Powder. Mix and dissolve it with Ammonium Molybdate Diluent (2×) and deionized water at a ratio of 12 mg : 10 mL : 10 mL (Powder : 2× Diluent : H₂O). Alternatively, weigh 120 mg of Ammonium Molybdate Powder and add it to 100 mL of deionized water. After complete dissolution, aliquot into 10 mL or other appropriate volumes. Mix an aliquot with an equal volume of Ammonium Molybdate Diluent (2×) before use. Store at -20°C to extend the shelf life. The prepared Ammonium Molybdate Chromogenic Solution can be stored at 4°C for up to 2 weeks. Discard if the solution becomes turbid or the color darkens. Please note that this product provides more Ammonium Molybdate Powder than the required amount.
4. Pi Loading
Set up blank tubes, standard tubes, and sample tubes according to the table below. Add solutions in the specified order, avoiding bubble formation, and mix gently. If the inorganic phosphorus concentration in the sample is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up duplicate tubes for samples and use the average value.
Component (mL) | Blank Tube | Standard Tube | Sample Tube |
ddH₂O | 0.05 | — | — |
Phosphorus Std. Working Sol. (25 μg/mL) | — | 0.05 | — |
Sample | — | — | 0.05 |
Ammonium Molybdate Chromogenic Sol. | 2 | 2 | 2 |
5. Pi Measurement
Mix well and incubate at room temperature for 5 minutes. Using a cuvette with a 1-cm light path, measure the absorbance at 340 nm on the spectrophotometer. Use the blank tube to zero the instrument. Record the absorbance values for the standard tube (A<sub>standard</sub>) and the sample tube (A<sub>sample</sub>).
6. Calculation 6.1 Formula for Inorganic Phosphorus in Serum/Plasma:
Pi (mmol/L) = (Asample / Astandard) × 0.807 × N
Pi (mg/L) = (Asample / Astandard) × 25 × N
6.2 Formula for Inorganic Phosphorus in Urine:
Pi (mmol/L) = (Asample / Astandard) × 0.807 × N
Pi (mg/L) = (Asample / Astandard) × 25 × N
6.3 Formula for Inorganic Phosphorus in Tissues:
Pi (mmol/mg prot) = (Asample / Astandard) × 0.807 × N / c
Parameter Description:
Asample: Absorbance of the sample tube
Astandard: Absorbance of the standard tube
N: Sample dilution factor
c: Protein concentration of the sample (mg/L)
Unit Conversion:
1 mg/dL = 10 mg/L = 1 mmol/L × 0.323
Reference Interval
Serum phosphorus concentration in healthy adults: 0.9 – 1.34 mmol/L (2.76 – 4.16 mg/dL)
Precautions
Assays should be performed within 5 minutes to 2 hours. The absorbance of the standard tube remains unchanged after 3 hours, while the absorbance of the sample tube increases over time.
Hemolyzed samples interfere with the detection; avoid using them whenever possible.
For icteric or lipemic samples, a sample blank should be prepared.
This method can be adapted for endpoint detection on automated biochemical analyzers.
The prepared Ammonium Molybdate Chromogenic Solution can be stored at 4°C for up to 2 weeks. Discard if the solution becomes turbid or the color darkens.
If the sample concentration is too high, dilute with distilled water and repeat the assay. Multiply the result by the dilution factor.
The absorbance measured at 340 nm is 82% of that measured at 325 nm.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Appendix
Standard Curve Preparation: Under room temperature conditions, follow the instructions and measure the absorbance at 340 nm for a series of phosphorus standards (5, 10, 15, 20, 25, 30, 35, 40 μg/mL) using a UV spectrophotometer. A sample standard curve is shown below (for reference only):
| I1510435 | Component | 100T | Storage |
| I1510435A | 磷标准 (1mg/mL) | 1 mL | 2-8℃. |
| I1510435B | 磷标准稀释液 | 2 mL | RT. |
| I1510435C | 钼酸铵粉末 | 1 EA | RT. |
| I1510435D | 钼酸铵稀释液 (2×) | 100 mL | RT. |
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