基於LC振盪器的介電係數生物感測器在食品安全牛奶微生物濃度的量測

Abstract

由於外食的族群越來越多，食品安全的議題一直是人們關注的焦點，這議題不僅影響著人們的生理及健康，也可能對整個社會和經濟產生重大影響，所以有需要發展快速簡便的生物感測器已應用在對於食品的品質感測。 本研究利用實驗室團隊基於LC振盪器原理所設計完成的微型積體電路雙參數介電係數感測器系統的，透過指叉型電極產生特定振盪頻率的電磁波，在近場與待測物質交互作用後，伴隨著對應介電係數實部的頻率變化與虛部的能量吸收衰減和波的量測，用以界定待測物質的變化情形。研究中以市售牛奶樣品(全脂3.0%~3.8%、低脂0.5%~1.5%)與可能因微生物濃度變化引起的特性變化，首先進行量化牛奶在室溫下隨存放時間(一天與七天)的變化情形，接著驗證在不同起始乳酸菌體積濃度下變化的情形，後續則以不同比例的酵母菌與乳酸菌進行牛乳中微生物交互作用的時間動態量測，最後則以純化的天然菌素(Nisin)進行可能機制的驗證。實驗數據顯示，這非接觸型介電係數感測器在空氣(n_air = 1)與純水(n_H2O = 1.33)的校正實驗中的讀值分別為(928.49±1.03E-02 MHz, 210.703±0.035 mV, N=3)與(880.99±1.50E-02 MHz, 213.148±0.046 mV, N=3)。在針對不同濃度的甲醇與乙醇量測中也成功在相平面上可以斜率差異分辨不同的物質濃度分佈。在牛奶的一天八小時的量測發現讀值斜率變化約為(全脂:-0.9265;低脂:-0.6156)，而在七天連續量測中不僅每天都有類似的斜率變化，在第五天之後斜率變化更為顯著(1-4天:-1.6384; 5-7天:-3.84)。而在加入不同濃度比例(1:2:4)乳酸菌時，在第一天的變化斜率(全脂1:4.12:8.767;低脂則為1:2.11:3.70)也呈現等比例遞增的現象，顯示微生物起始濃度對於牛奶的反應速度是有等比例影響。在加入不同濃度比例的酵母菌時則能由量測數值變化觀察出兩種類別微生物之間的交互作用的動態差異，不僅在斜率上有改變的趨勢，也可以在轉折點上反應出作用時間的提前或延後。藉加入純化的天然菌素Nisin，可以了解不同微生物之間可能的拮抗作用機制。 本論文透過指叉型電極在物質中電磁波的傳播伴隨著能量吸收和波的相位變化的積體化電路量測，成功檢測牛奶中因微生物濃度增加引起的共振頻率變化與振幅增加與衰減，這有助於未來進一步識別樣品中細菌生長濃度的變化的原因，可望透過待測物性質的標準數據或相對變化區間範圍，提供消費者存放過程中的控制參考。透過測量微生物含量，及早檢測微生物的增殖，有助於預防牛乳的過早變質以及適當的保存下仍可以在不危害人體健康下有效食用，以減少食物之浪費。

With the increasing number of people dining out, food safety has become a focal point of public concern. This issue not only affects individuals' health but also has significant societal and economic impacts. Therefore, the development of rapid and convenient biosensors for food quality sensing is essential. This study utilizes a dual-parameter dielectric constant sensor system based on an LC oscillator principle, designed and developed by our laboratory team. This system employs interdigital electrodes to generate electromagnetic waves at specific oscillation frequencies. When these waves interact with the test substance in the near field, changes in the dielectric constant's real part cause frequency shifts, and energy absorption and wave attenuation reflect changes in the imaginary part. This study uses commercially available milk samples (whole milk 3.0%-3.8%, low-fat 0.5%-1.5%) and examines changes in their properties due to variations in microbial concentrations. Initially, we quantified changes in milk stored at room temperature over time (one day and seven days). We then validated the changes at different initial concentrations of lactic acid bacteria. Subsequently, we conducted dynamic measurements of microbial interactions in milk with varying ratios of yeast and lactic acid bacteria. Finally, we used purified natural bacteriocin (Nisin) to verify possible mechanisms. Experimental results showed that this non-contact dielectric constant sensor had readings of (928.49±1.03E-02 MHz, 210.703±0.035 mV, N=3) in air (n_air = 1) and (880.99±1.50E-02 MHz, 213.148±0.046 mV) in pure water (n_H2O = 1.33) in calibration experiments. The sensor successfully distinguished different concentrations of methanol and ethanol based on slope differences in the phase plane. During an eight-hour measurement of milk over one day, the slope changes were approximately (whole milk:-0.9265;low-fat:-0.6156). In seven-day continuous measurements, similar slope changes were observed daily, with more significant changes after the fifth day (day 1-4:-1.6384; day 5-7:-3.84). When different concentrations of lactic acid bacteria were added (1:2:4), the first day's slope changes (whole milk 1:4.12:8.767; low-fat milk 1:2.11:3.70) showed a proportional increase, indicating that the initial concentration of microorganisms proportionally affects the reaction speed in milk. When different concentrations of yeast were added, the dynamic interaction differences between the two types of microorganisms were observed through measurement value changes. This not only altered the slope trend but also reflected shifts in the action time points. The addition of purified natural bacteriocin Nisin helped understand the possible antagonistic mechanisms between different microorganisms. This study successfully detected changes in resonance frequency and amplitude attenuation in milk due to increased microbial concentrations through an integrated circuit measurement using interdigital electrodes to transmit electromagnetic waves accompanied by energy absorption and phase changes. This can aid in further identifying the reasons for changes in bacterial growth concentrations in samples. Establishing standard data or relative variation ranges for the properties of the test substances can provide consumers with control references during storage. Early detection of microbial proliferation through measuring microbial content helps prevent premature spoilage of milk, ensuring safe consumption under proper storage conditions, and reducing food waste.