DNA hydrogels combined with microfluidic chips for melamine detection

Highlights

• A specific colorimetric detection method for melamine using AuNPs - encapsulated DNA hydrogel was constructed.

• A POCT detection platform is prepared by combining microfluidic chip.

• The POCT platform can detect melamine with a detection limit to 37 nM.

• By changing the aptamer in DNA hydrogel, this strategy can be used to detect other targets.

Abstract

The illegal addition of melamine (MEL) into pet foods and infant milk powder has caused great concern among people. In this study, a point-of-care testing (POCT) method was developed by combining stimuli-responsive deoxyribonucleic acid (DNA) hydrogels with microfluidic chips to achieve portable and sensitive detection of MEL. With the MEL aptamer (MA) acting as a cross-linker, DNA hydrogel-coated gold nanoparticles (AuNPs) served as a basis for colorimetric detection and quantitative analysis. In the presence of MEL, it competitively binds to the aptamer, causing disintegration of the DNA hydrogels and a release of the coated AuNPs, making it possible to visually detect and quantitatively measure the MEL. Under optimal conditions, the detection range of MEL using DNA hydrogels was 0.1–100 μM and the limit of detection (LOD) was 42 nM. This portable, sensitive, and user-friendly field test equipment was developed to avoid the use of non-portable laboratory instruments. Furthermore, we combined microfluidic chips with the properties of DNA hydrogels, making it possible to quantitatively detect MEL by taking photos and analyzing the gray value using software in accordance with the different colors of copolymerization solutions after the reaction. The detection range of MEL using the microfluidic chip-based method was 0.2–50 μM, and the LOD was 37 nM. Neither trained operators nor large-scale instruments are needed for using this method to conduct portable and sensitive field detection of the targets, which highlights the methods excellent potential in food security, clinical tests, environmental monitoring, and other aspects.

Introduction

Melamine (MEL) is one of the raw materials used for the production of plastics, coatings, and adhesives [1]. Owing to its high nitrogen content, MEL is added to dairy products and animal feed to increase the apparent protein content [2] as determined by the Kjeldahl method that is usually adopted to determine the protein content in foods or feed. The illegal addition of MEL led to pet food contamination in the United States in 2007 and infant milk powder adulteration in China in 2008 [3], Research indicates that intake of MEL causes lithiasis and other illnesses in animals and infants [4,5]. MEL is classified as a group 2B carcinogen by the International Agency for Research on Cancer (IARC). United Nations Food Standards Commission set a safe limit of MEL in foods at 2.5 mg L⁻¹ (19.8 μM) and in infant milk powder at 1 mg L⁻¹ (7.9 μM), and therefore, MEL detection has attracted increasing attention from researchers.

Traditional MEL detection methods, including liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, and atomic absorption spectrographic method [6] require expensive large-scale instruments, strict pretreatment of samples, and highly skilled operators. With the advantages of high sensitivity, low limit of detection (LOD), and simple processing, nanotechnology-based detection methods have greatly helped to advance the development of rapid detection technologies to overcome the shortcomings of traditional technologies in MEL detection.

Hazra et al. achieved sensitive detection of MEL based on the selective quenching of the luminescence signal of upconversion nanocrystals by melamine [7]. Liao et al. opened and closed the fluorescence signals of β-cyclodextrin modified carbon nanoparticles by replacing Fe³⁺ with MEL [8] and obtained outstanding results for MEL detection in milk. Xie et al. prepared microfluidic paper-based analytical devices (μPADs) based on gold nanoparticles (AuNPs) [9] to implement a low-cost, portable, and sensitive method for the detection of MEL, which is therefore highly suitable for use in regions with limited resources. Among the various detection methods, point-of-care testing (POCT) methods has the advantages of fast detection, simple use and saving comprehensive cost appeal to the author. Common POCT technologies include lateral flow immunoassays (such as virus test paper [10]), sensor technology (such as glucometer [11]), and microfluidic technology [12], which have grown rapidly in recent years. Microfluidic chips have been widely applied to POCT detection platforms owing to their advantages of low cost and transformation of different signals [[13], [14], [15], [16]]. Compared to conventional quantitative analysis, colorimetric detection in POCT allows for both real-time qualitative and semiquantitative analyses without relying on any advanced or complex instruments. It is worth noting that AuNPs have been favored by researchers in the application of colorimetric detection for a long time because of their mature synthetic technology and stability [17]. To achieve the goal of both colorimetric and quantitative detection and develop a user-friendly POCT detection platform, deoxyribonucleic acid (DNA) hydrogels were combined with microfluidic chips in this study. As a result, colorimetric detection was possible based on the color of the copolymerization solutions after the reaction, and quantification could also be carried out simply by analyzing the gray value with specific software instead of using large-scale instruments.

Stimuli-responsive DNA hydrogels possess both the programmability of DNAs and the good bearing capacity of hydrogels [18], and therefore receive increasing attention in the development of biosensors. The rational design and precise application of DNA strands enable hydrogels to have greater responsiveness, including responsiveness to non-biological (light [19], heat [20], magnetism [21], pH [22], etc.) and biological stimuli (nucleic acids [23], proteins [24], small molecules [25], etc.). Generally, a specific response to the target can be achieved by introducing the nucleic acid aptamer into hydrogels. The nucleic acid aptamer can be separated using systematic evolution of ligands by exponential enrichment (SELEX) technology, and the nucleic acid aptamer have the advantages of a wide target range, massive scale in vitro synthesis, great repeatability, high stability, and low cost [26]. The MEL aptamer (MA) adopted in this study was modified from the aptamer obtained by Gu et al. using SELEX [27], and displayed good affinity and better selectivity. This detection platform has potential for further application in the portable and quantitative detection of other targets by altering the aptamer used in the system.

In this study, a portable and sensitive POCT detection platform was designed, in which aptamer-based stimuli-responsive DNA hydrogels are combined with microfluidic chips, enabling the sensitive detection of MEL. First, AuNPs were encapsulated in polyacrylamide DNA hydrogels bridged by MA. Next, these complexes competitively bind to the aptamer in the presence of MEL, which leads to hydrogel rupture and AuNPs release. The AuNPs used in this method provide a basis for the colorimetric and quantitative detection of the targets. In this case, microfluidic chips were introduced to construct a POTC detecting platform for immediate analysis at the sampling site. The DNA hydrogels did not flow directly from the reaction area to the lower detection area, however, following the completion of the reaction, the copolymerization solutions did flow into the detection area under gravity. At that time, photos were taken using mobile phones and the gray value was analyzed using ImageJ software to perform a quantitative analysis of the detected concentrations. The target concentration was positively correlated with the amount of AuNPs released by the DNA hydrogels and negatively correlated with the gray value. The POCT detection equipment showed a lower LOD of 37 nM for MEL. As a result of this study, we developed a portable, inexpensive, and simple to operate detection platform for quantitative analysis, which does not depend on large-scale laboratory instruments.