Foreword
In molecular biology experiments, the accurate detection of nucleic acid concentration and purity is a core prerequisite for the success of subsequent experiments such as PCR and gene sequencing. Excessively high concentrations may inhibit the reaction system, while excessively low concentrations can affect experimental sensitivity and even lead to direct experimental failure.

Currently, the mainstream methods for nucleic acid concentration detection are absorbance and fluorescence methods, with corresponding instruments including micro-spectrophotometers, fluorometers, and fluorescence microplate readers, respectively. Although they all output concentration values, their detection principles differ significantly, and they are suited to different application scenarios.

Today we'll take a comprehensive look at how to choose the right instruments for different scenarios and avoid common testing mistakes.


01 Differences in Detection Principles
● Micro-Spectrophotometer: Detection Based on the Physical Properties of Nucleic Acids

It’s the most basic and commonly used nucleic acid concentration detection instrument. Its core principle is "the ultraviolet absorption characteristics of nucleic acids" - purine and pyrimidine bases in nucleic acid molecules specifically absorb ultraviolet light at a wavelength of 260 nm, while proteins (mainly interfering impurities) absorb ultraviolet light at a wavelength of 280 nm. The instrument detects the absorbance value (A260) at 260 nm, according to the Lambert-Beer Law:

A = kbc, where:

A represents absorbance, which indicates the degree to which the intensity of light decreases after passing through the solution;

k is a proportionality constant, which is related to the wavelength of the incident light, the properties of the solute, and the properties of the solvent;

b represents the liquid layer thickness, i.e., the optical path length, usually expressed in centimeters (cm).

c is the concentration of the light-absorbing substance in the solution, usually expressed in moles per liter (mol/L) or grams per liter (g/L).

By combining fixed conversion formulas (such as 1 A260 = 50 μg/mL dsDNA, 40 μg/mL RNA), the nucleic acid concentration can be quickly calculated; at the same time, the purity of nucleic acid can be judged by the ratio of A260/A280 (ideal range 1.8-2.0) (a deviation in the ratio indicates protein contamination).

In short: it doesn't rely on any additional reagents, directly "seeing" the absorbance of the nucleic acid itself, and quickly producing results. However, since all types of nucleic acids have absorbance values at 260 nm, it cannot distinguish between nucleic acid types, and the results are not very accurate, but it can indicate the purity of the sample.


● Fluorometer, Fluorescence Microplate Reader: Marked with Fluorescent Dyes
The core principle of a fluorometer is to use light of a specific wavelength to excite fluorescent substances in a sample, and then calculate the concentration of the target substance by measuring the intensity of the emitted fluorescence. It needs to be used with a specific fluorescent dye - this type of dye specifically binds to nucleic acid molecules. For example, the fluorescent dye in the dsDNA detection reagents only binds to dsDNA, not ssDNA or RNA.

When the dye is not bound to nucleic acid, the fluorescence signal is extremely weak; once it binds to nucleic acid, it is activated by excitation light of a specific wavelength, releasing a strong fluorescence signal, and the fluorescence intensity is linearly positively correlated with the nucleic acid concentration. The instrument can accurately calculate the nucleic acid concentration by detecting the fluorescence intensity.

Simply put: it needs a "helper" (fluorescent dye) to identify only nucleic acids, avoid interference from impurities, and make the detection more accurate. However, it cannot indicate sample purity information.

The detection principle of a fluorescence microplate reader is the same as that of a fluorometer, but it has a higher throughput.


02 Advantages and Disadvantages in Common Molecular Detection Methods
We combine PCR testing and NGS (next-generation sequencing), two of the most commonly used scenarios, to analyze the compatibility, advantages and disadvantages of the three types of instruments one by one, so that everyone can choose the testing method according to the application.
 


The requirements for nucleic acid concentration in PCR testing are "rapid screening and meeting purity standards". There is no need to excessively pursue ultra-high sensitivity. The key is to eliminate the inhibition of PCR reaction by impurities such as proteins.

● Micro-Spectrophotometer
Advantages: Fast detection speed (5s/sample), no reagents required, low cost, can simultaneously determine purity (A260/A280), suitable for rapid screening of PCR samples; simple operation, no pre-incubation required, even beginners can quickly get started.

Disadvantages: Low sensitivity (minimum detection concentration approximately 2 ng/μL), unable to distinguish nucleic acid types (dsDNA, ssRNA, degraded nucleic acids cannot be distinguished); impurities such as phenols and salt ions in the sample will interfere with absorbance detection, leading to inaccurate results.

● Fluorometer, Fluorescence Microplate Reader
Advantages: Higher sensitivity than micro-spectrophotometers (minimum detection concentration approximately 0.1 pg/μL), high specificity, unaffected by impurities such as proteins and salt ions, resulting in more accurate results. With appropriate dyes, it can precisely distinguish nucleic acid types.

Disadvantages: Only provide concentration values, not sample purity information. Require precise sample addition and high operational skill. Require the use of fluorescent dyes during detection, increasing costs. Detection speed is relatively slow (requires 2-3 minutes of incubation).



NGS requires high accuracy, good repeatability, and no interference from impurities in nucleic acid concentration and purity. Sequencing samples are often processed in batches, and sometimes the original nucleic acid concentration of the sample is below 1 ng/μL. The accuracy of sample concentration is extremely important, otherwise it will affect the sequencing quality and data accuracy.

● Micro-Spectrophotometer
Advantages: Quickly screens sample purity, eliminating obvious impurities; no reagent costs, suitable for preliminary screening of NGS samples. Remove samples with extremely poor purity.

Disadvantages: Insufficient sensitivity, unable to accurately detect the low concentration of nucleic acids required by NGS; unable to distinguish degraded nucleic acids (NGS has high requirements for nucleic acid integrity, and degraded nucleic acids will affect sequencing results, but micro-spectrophotometers cannot identify them); results repeatability is generally poor, making it unsuitable for final concentration quantification of NGS samples.

● Fluorometer
Advantages: High sensitivity, accurately detecting low concentrations of nucleic acids required for NGS; high specificity, unaffected by impurities, and good repeatability.

Disadvantages: Low throughput, only 1-8 samples can be tested at a time, unsuitable for batch NGS sample detection; high dye cost, resulting in higher expenses for batch detection.

● Fluorescence Microplate Reader
Advantages: Perfectly suited for NGS batch detection needs; high throughput (96-well plate) allows for rapid concentration quantification of hundreds of samples; high sensitivity and specificity, excellent result repeatability, and precise control of nucleic acid concentration within the required NGS range.

Disadvantages: High instrument cost (suitable for laboratories with batch sequencing needs); expensive reagents; complex operation; require professional maintenance and operation.


Therefore, save this table to master the selection of three types of instruments.




Final Supplement: Key Selection Recommendations
If the laboratory mainly uses PCR and small-sample testing, and pursues low cost and high efficiency, a "micro-spectrophotometer" is sufficient to meet basic daily needs.

If low concentrations of nucleic acids or micro samples (such as cfDNA) are involved, and the sample volume is small, choose a "fluorometer" to balance sensitivity and accuracy.

If a laboratory has batch testing needs (such as NGS, clinical batch samples) and pursues high accuracy and high throughput, it is advisable to choose a "fluorescence microplate reader". Although the cost is high, it can improve experimental efficiency and result stability.