Application of HEPES Sodium Salt in Flow Fluorescence Luminescence Test Kit (Part 1)

HEPES sodium salt (CAS number 75277-39-3), chemical name N - (2-hydroxyethyl) piperazine-N '- (2-ethylsulfonic acid) sodium salt, molecular formula C ₈ H ₁ N ₂ NaO ₄ S, is a high-quality zwitterionic biological buffering agent. HEPES sodium salt belongs to the Good's buffer family, with a pKa of 7.5 at 25 ℃ and an effective buffering range of pH 6.8-8.2. This buffer range precisely covers the pH range required for the vast majority of physiological and in vitro diagnostic reactions, making it a core component of buffer systems widely used in fields such as cell culture, biopharmaceuticals, and diagnostic reagents.

HEPES sodium salt has multiple unique physicochemical advantages: it has extremely low binding ability to metal ions and does not bind to divalent metal ions such as magnesium, calcium, and manganese; High optical transparency, extremely low absorption in visible and ultraviolet regions, does not interfere with fluorescence detection; Low cytotoxicity, superior to traditional buffering agents such as Tris and phosphate; Good temperature stability and minimal pH variation with temperature. These characteristics give HEPES sodium salt an irreplaceable position in in vitro diagnostic kits involving fluorescence detection.

Introduction to Flow Fluorescence Luminescence Method

Flow Fluorescence Luminescence Assay is a rapidly developing multi index joint detection technique in the field of in vitro diagnostics (IVD) in recent years. Compared with traditional chemiluminescence single item detection, flow cytometry fluorescence detection can simultaneously detect multiple items and has the advantage of high throughput. During the detection process, the stability of the buffer system directly determines the efficiency of the antigen antibody reaction, the dispersion state of the microspheres, and the intensity and reproducibility of the fluorescence signal.

Specific application of HEPES sodium salt in flow cytometry fluorescence assay kit

Currently, multiple approved flow cytometry detection kits use HEPES sodium salt buffer as the core component. Here are two representative examples:

Squamous cell carcinoma antigen detection kit (flow cytometry fluorescence luminescence method):

In the composition of the kit, the microsphere coated antibody reagent (A) includes microspheres coated with SCCA (mouse) antibody and HEPES sodium salt buffer; The biotin antibody reagent (B) contains biotinylated SCCA (mouse) antibodies and HEPES sodium salt buffer. The product needs to be stored in the dark at 2-8 ℃ and has a shelf life of 12 months.

Cytokine detection kit (flow cytometry fluorescence luminescence method):

This kit is mainly used clinically to monitor the immune status and inflammatory response of the body. HEPES sodium salt buffer is used in multiple components: Microsphere coated antibody reagent (A) contains six types of coated antibody microspheres and HEPES sodium salt buffer; The biotin antibody reagent (B) contains six biotinylated antibodies and HEPES sodium salt buffer; The calibration samples (CAL2-CAL6) and quality control samples (QCL, QCH) are also based on HEPES sodium salt buffer.

Typical formula of HEPES sodium salt in buffer solution

In practical applications, HEPES sodium salt is often used in combination with other components to construct a complete buffer system. The buffer used for flow cytometry fluorescence detection usually includes a base buffer, a surfactant, and a preservative; The basic buffer includes one or more of Tris buffer, MES buffer, CBS buffer, PBS buffer, and HEPES buffer. The concentration of solutes in the basic buffer is generally 50-100 mM, and the pH value is 7.3-7.5.

In the classic applications of flow cytometry and fluorescence detection, common formulations of HEPES buffer include: 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 7.0; Or 10mM HEPES, 136mM NaCl, 2.7mM KCl, 2mM MgCl ₂, 5.5mM glucose, 0.1% BSA, pH 7.35.

Starting from the physicochemical properties of HEPES sodium salt, this article systematically introduces its specific application scenarios in flow cytometry fluorescence assay kits. The next article will delve into the core technological advantages of HEPES sodium salt in flow cytometry fluorescence detection from the dimensions of pH buffer range, metal ion chelation, optical transparency, chemical inertness, and cytotoxicity. Based on the measured indicators of New Desheng HEPES sodium salt products, key parameters and procurement suggestions for raw material selection will be provided, providing reference for raw material selection and quality control of reagent kit production enterprises.