Optical Biosensor for the Detection of Hydrogen Peroxide in Milk ^†

Abstract

Over the years, the food industry’s concern to provide safe food that does not cause harm or illness to consumers has increased. The growing demand for the detection of compounds that can contaminate food is increasingly important. Hydrogen peroxide is frequently used as a substance to control the growth of microorganisms in milk, thus increasing its shelf life. Here, a strategy is presented for the detection of hydrogen peroxide as a milk adulterant, using a single shot membrane sensor. The lowest concentration measured with this technique was 0.002% w/w of H₂O₂ in semi-fat milk.

1. Introduction

Milk is one of the most complete foods for humans, containing nutrients including carbohydrates, proteins, fats, minerals, and vitamins [1].

Owing to its rich composition, milk becomes a substrate for the growth of undesirable microorganisms that can easily deteriorate the product. To prevent this from happening, prohibited substances are fraudulently added [2]. Hydrogen peroxide (H₂O₂), hypochlorite, formaldehyde, potassium dichromate, and salicylic acid are examples of substances used as adulterants that need monitoring and quality control as they are toxic to humans [3].

In the case of H₂O₂, it is widely used in the dairy industry as an antimicrobial agent, thus helping to preserve raw milk in the absence of refrigeration [4]. Despite its conventional use, when added to milk, H₂O₂ can cause a decrease in the nutritional value of the food due to the destruction of vitamins A and E, which generate reactive and cytotoxic oxygen species, including hydroxyl radicals, that can initiate oxidation and damage nucleic acids, lipids, and proteins. Consequently, when ingested, milk can lead to negative effects on the health of the population, especially in immunocompromised people [2,4].

In the USA, hydrogen peroxide is used in cheese production in concentrations up to 0.05% w/w, however, in other countries, its addition is prohibited due to its toxic effects. A peroxide concentration > 0.1% w/w has been proven to induce cancer in the duodenum of mice and present short-term genotoxicity [3].

Here, a study is presented for the detection and quantification of H₂O₂ using a chemiluminescence technique. A small, low-cost hydroxyethyl cellulose sensitive membrane combined with a high-sensitive photodetector is used to measure H₂O₂ concentrations in semi-fat milk samples.

2. Materials and Methods

The sensing methodology is based on the detection of a luminescence signal from the chemical reaction within a solid membrane produced with hydroxyethyl cellulose (HEC, Sigma Aldrich, Taufkirchen, Germany), luminol, sodium phosphate, cobalt (II) chloride hexahydrate, sodium lauryl sulphate (SLS), and ethylenediaminetetraacetic acid (EDTA).

The procedure established by Omanovic-Miklicanin [5] was refined to establish experimental protocols. For the determination of H₂O₂ in very low concentrations, the sensor sensitivity should be as high as possible. Therefore, the systematic optimization of the membrane was necessary. Only one constituent was varied at a time, keeping the remaining constituents unchanged. After membrane optimization, the final concentrations of these constituents were set to luminol (0.2 mg), sodium phosphate (8.6 mg), SLS (60 μL, 34.36 mmol/L), cobalt hydroxide (100 μL, 5.0 mmol/L), EDTA (2 µL, 20 µmol/L), and HEC (150 mg) was added to 10 mL of Milli-Q^® water.

The membrane solution was placed on a magnetic stirrer for 30 min. Individual 3D printed cups were used, and 1000 µL of membrane solution was added and dried for 4 h (T = 70 °C). After drying, the membranes were stored in a desiccator under a vacuum. For the measurement procedure, the membrane was placed directly onto the membrane holder on top of the detector. The light emission was measured by adding 500 µL of the sample solution.

For straight and rapid spectrophotometric H₂O₂ detection, a detection module was built containing a highly sensitive detection system with a photodiode (model S8746-01 Hamamatsu Japan), a dedicated amplification system with variable gain, and an embed controlling unit. The sensitive optoelectronic system was isolated inside of a custom-made 3D printed case allowing the easy replacement of the sensing membrane and allowing the sample pipetting, preventing the detection of the ambient light. This module was powered with a low noise power source, and the data was acquired and analyzed with a user-friendly graphical interface (GUI) and a raspberry pi (Figure 1).

3. Results and Discussion

Semi-fat milk samples were adulterated with H₂O₂ concentrations from 0.001% w/w to 0.006% w/w by diluting a standard 30% w/w solution of H₂O₂. The variation of the chemiluminescent intensity is presented in Figure 2 for all samples, together with the time integral of the decaying chemiluminescent signal for each H₂O₂ concentration.

Taking into consideration that 0.05 % w/w of H₂O₂ is the defined limit for the FDA in milk for cheese production [6], the developed sensor would be suitable for determinations of H₂O₂ as a fraud controller in milk samples within the legal limits of different countries. Moreover, to achieve a more practical approach to the commonly time-consuming sample preparation methods, the pre-treatment step was successfully eliminated. In fact, the optimized sensor requires minimal solvent use and waste production. When compared with other methods available for the determination of H₂O₂ presence in milk, this portable biosensor is an easy and reliable method that ensures the required sensitivity while offering a low time of analysis and no need for additional laboratory equipment.

The methodology developed and optimized demonstrates that it is possible to detect very low concentrations of H₂O₂ (down to 0.001 % w/w in an aqueous system). As the H₂O₂ concentration increased, the intensity of the emitted light and the reaction time increased. Low limits of detection were achieved, thus indicating the applicability of this assay to real samples exhibiting the required sensitivity for the analytical determination of H₂O₂ in biological samples such as milk.

In this work, the reaction of H₂O₂ and luminol catalyzed by cobalt hydroxide was used to detect H₂O₂ in milk; however, another spectrophotometric method was described by Lima et al. [2] for the detection of H₂O₂ in milk, using the reaction between hydrogen peroxide and guaiacol, catalyzed by peroxidase, producing a red product, where a low detection limit was obtained.

4. Conclusions

The proposed sensor provided to be a rapid, cost-effective, and environmentally friendly approach for the determination of hydrogen peroxide as a milk adulterant. This optimized and validated method has a very good linearity range when the sample is in its liquid state, where concentrations of H₂O₂ as low as 0.001% w/w can be detected with good repeatability. As a practical application for this methodology under controlled conditions, an adulterated milk sample was analyzed. Concentrations of H₂O₂ of 0.002% w/w to 0.006% were detected, and the method was calibrated for semi-fat milk, proving that the limit of detection and linearity range of the proposed method are suitable for the analysis of milk samples in loco, which can add value to the food fraud department. Moreover, the reagents required are commonly used in analytical laboratories, are inexpensive, and can be consumed in low amounts (500 µL), thus resulting in negligible and non-toxic waste generation. In addition to the mentioned advantageous features, the proposed method validation is comparable to those found in the literature.