1. Introduction
Biogenic amines (BAs) are important compounds that can determine the quality of food [
1]. High levels of amines in food are produced by bacterial decarboxylation of amino acids and have been recognized as an important reason for seafood intoxication [
2]. Hence, the determination of the concentration of BAs in fresh food is in high need to determine its freshness status. Papageorgiou and co-workers gave a review on food and beverage products that should be regularly tested since they contain BAs or may develop certain contents over (storage) time [
3]. This raises interest in research on rapid and inexpensive optical detection methods and tools like dipsticks to determine BAs in food, not only for individual concentration levels but also as a sum parameter. According to the European Food Safety Authority (EFSA), the U.S. Food and Drug Administration (FDA) as well as the World Health Organization (WHO), there are limits for BA concentrations in food to control the food quality. Histamine is one of the most bioactive and toxic BAs. Histamine exists in the majority of foods and plays an important role in food intolerances [
4]. If the histamine concentration in food exceeds 500 mg/kg, there is a high risk for food poisoning [
3]. In addition, 5–10 mg of histamine might induce skin irritation, rashes, dilatation of peripheral blood vessels resulting in hypotension and headache, or contractions of intestinal smooth muscles causing diarrhea and vomiting [
5]. Furthermore, 10 mg is regarded as a borderline for toxicity and 100 mg can result in medium toxic responses, and 1000 mg is considered as very toxic [
3].
Optical detection of BAs is challenging because those are weak absorbers of visible light as most of them lack conjugated aromatic π-electron systems. The solution to this problem can be labeling or derivatization of BAs with chromophores or fluorophores. Additionally, the analyte has to be extracted from out of a complex matrix in food analysis. Therefore, the combination of separation techniques such as GC, HPLC, or capillary electrophoresis with optical, electrochemical, or mass spectrometric detection after BA derivatization was used recently [
6,
7,
8,
9,
10,
11]. Eventually, ELISAs [
12] are used for BA determination in food samples. Those are highly selective and sensitive on the one hand but expensive, time-consuming, and require highly trained staff, on the other hand. In order to overcome these limitations, reasonably fast, low-cost, and portable chemo and biosensors are desired for rapid on-site analysis of BAs in food [
13].
Frequently, chromogenic and fluorogenic dyes, such as acid-base indicators [
14,
15,
16] porphyrins [
17], phthalocyanines [
18], chameleon dyes [
19,
20], coumarin derivatives [
21], azo dyes [
22,
23], or nanomaterials are applied for optical BA determination. Cellulose-based microparticles bonded with a pH-indicator and a blue reference dye yielded in slow traffic light-responding (1.5 h) colorimetric sensors [
16]. A very recent concept combined two layers for BA sensing, one with colorimetry and one for laser desorption ionization mass spectrometry [
24]. Another study described the highly specific and sensitive detection of histamine in mackerel using thin-layer chromatography with visualization by spraying the sheets with ninhydrin and diazonium reagents [
25]. Furthermore, an array of five pH-indicators was shown to respond quickly (10 min) and to differentiate between isobutylamine, triethylamine, and isopentylamine in ppm concentrations via RGB readout using a mobile phone [
15]. Mobile phones have become increasingly popular in food sensing in recent years [
26]. In most cases, array sensors require additional chemometric data treatment to decipher the individual BA and its concentration from the response received by multiple receptors of low selectivity SEM images [
15,
27]. Unlike arrays, dipsticks only need a one-point readout and are therefore much quicker and easier to be read out and thus deliver their response much faster. The development of dipsticks is beneficial since they are practical, simple, portable, easy to use, and thus do not require trained staff. Moreover, they have a much lower cost compared to instrumental methods of analysis [
14]. In addition to that, colorimetric sensing of BAs using dipsticks provides a simple response based on the color change, and this leads to a yes/no answer besides the quantitative analysis [
19,
28].
Direct sensing of BAs was recently carried out using filter paper-based dipsticks containing an amine-reactive chromogenic probe and a reference dye. Quantitative determination of BAs could be successfully achieved either visually based on a color change or via luminescence. Digital images of the luminescence of sensor spots were taken, and the BA concentrations were derived from the red-to-green intensity ratio via ImageJ software [
19]. As an alternative sensor concept to paper as support for the hydrogel carrying the sensing matrix, electrospun nanofibers on ITO sheets were employed. Dipsticks containing these nanofiber mats showed an up to six-fold higher sensitivity compared to those based on hydrogel sensor membranes containing the same dye [
29]. This is due to the high surface area to volume ratio and the high porosity of electrospun nanofibers. Moreover, electrospun nanofibers were designed such that they were counter-charged with respect to BAs in order to achieve an additional enrichment effect.
Reflectometric detection of optical sensors raises interest, since it is a fast and simple technique. Reflectometric sensors require a light source (which could be an inexpensive LED), a filter to select the detection wavelength and a detector (which can be a low-cost digital camera). Evaluation of data can then be carried out by free available software. Hence, a complete sensor (array) can be obtained for a few hundred US-
$ or less including detection equipment. BA sensing was carried out reflectometrically by a digital camera and data were analyzed using the color space of the International Commission on Illumination (CIE) system [
16]. These sensors were applied for quantitation of various amines and ammonia produced during food ageing in food packages. Fish freshness was monitored reflectometrically using a colorimetric sensor of creatine in fish, which is an indicator for the fish ageing [
30].
In order to create dipsticks for food analysis with improved features, the following novel features were implemented: (a) a sensor layer containing only one dye instead of two to simplify the dipsticks compared to earlier research [
29]; (b) using reflectometry for a one-step detection (instead of a two-step detection process as formerly required with digital photography evaluation [
19]); and (c) using an NIR chromogenic dye to reduce the effects of self-absorption and scatter upon measuring in real samples.
Therefore, for the first time, an NIR dye reactive to BAs was embedded into a mat of electrospun nanofibers to form reflectometric dipstick sensors. The electrospun nanofibers made from cellulose acetate (CA) are prepared by a simple standard electrospinning procedure and contain the S0378 cyanine dye which absorbs at 800 nm. CA is stable over a wide range of pH values and contains many hydroxyl (OH) and ester groups which render it highly hydrophilic. This allows easy access of polar analytes like BAs to the S0378 chemosensor embedded inside the NFs. Additionally, CA can be easily electrospun into layers of several tens of µm, as common in optical chemical sensors. Primary amines react with the dye by an SN1 nucleophilic substitution mechanism which is accompanied by a color change from green to blue. Hence, the concentration of BAs can be determined based on reflectance detection which is a simple and fast readout. The equal response towards monoamines and diamines makes the dipstick an ideal tool for determination of the total amine content (TAC) in real samples which was demonstrated by monitoring the ageing of shrimp samples over time.