Electrochemical DNA-Based Sensors for Molecular Quality Control: Continuous, Real-Time Melamine Detection in Flowing Whole Milk

Abstract

The ability to monitor specific molecules in real-time directly in a flowing sample stream and in a manner that does not adulterate that stream could greatly augment quality control in, for example, food processing and pharmaceutical manufacturing. Because they are continuous, reagentless, and able to work directly in complex samples, electrochemical DNA-based (E-DNA) sensors, a modular and, thus, general sensing platform, are promising candidates to fill this role. In support, we describe here an E-DNA sensor supporting the continuous, real-time measurement of melamine in flowing milk. Using target-driven DNA triplex formation to generate an electrochemical output, the sensor responds to rising and falling melamine concentration in seconds without contaminating the product stream. The continuous, autonomous, real-time operation of sensors such as this could provide unprecedented safety, convenience, and cost-effectiveness relative to the batch processes historically employed in molecular quality control.

Figure 1.

An electrochemical DNA-based (E-DNA) sensor supporting continuous, reagentless detection in a complex sample stream. Here, we have fabricated and validated sensors for the continuous monitoring of melamine (M) using a family of redox-reporter (methylene blue, MB) modified, thiol-anchored DNA sequences comprised of two polythymine segments connected by a four-cytosine loop: T_nC₄T_n, with the length of the polythymine varying from 4 to 12 bases. By tuning the length of polythymine segments, we expect that we could tune the extent of cooperativity.

Figure 2.

Sensors for the continuous, high frequency measurement of melamine in simple buffer solutions and in whole milk. (Top) When challenged with 500 μM melamine in a simple buffered solution (A), the T20 sensor’s signaling current changes dramatically and (B) the binding event is effectively complete within the few seconds required to perform the first square-wave scan. (Bottom) When challenged with the same amount of melamine in undiluted whole milk, we observe (C) a significant signal change and (D) rapid binding event. Of note, sensors built from the other two T-rich sequences we have explored exhibit similar binding kinetics (Figure S1). The error bars here and in the following figures reflect the standard deviation of measurements derived from at least three independently fabricated sensors, illustrating the magnitude of the sensor-to-sensor variation.

Figure 3.

The sensors are selective and specific. For example, the T20 sensor shown here (A) exhibits little loss in signal when transferred from simple buffer solution to undiluted whole milk and (B, C) performs well when challenged in this complex sample matrix. Specifically, the signal gain observed in undiluted whole milk is within the error of that seen in simple buffer, when challenging the sensors with 1 mM melamine. The sensor is also specific, responding to melamine but not (<5% signal change, which is within normal sensor-to-sensor variation for these hand-fabricated devices) to its molecular analogue, cyanuric acid. Of note, sensors built from the other two T-rich sequences we have explored exhibit similar selectivity (Figure S1).

Figure 4.

The sensors are quantitative. The response curves of sensors fabricated using T12, T20, and T28 in (A) simple buffer and (B) undiluted whole milk. Due to their thymine rich nature, these constructs are cooperative, steepening their binding curves. They are less cooperative, however, when challenged in milk rather than in a simpler sample matrix. The signals produced at lower concentrations (below 50 μM) are presented in Figure S3.

Figure 5.

Continuous, real-time molecular monitoring in a flowing sample stream. We challenged a melamine-detecting sensor in undiluted, flowing whole milk to demonstrate the real-time measurement of this target. Specifically, we fabricated these sensors with a T20 construct and challenged them against varying pulses of the target in flowing whole milk over the course of several hours. As expected, our sensors responded correspondingly and rapidly to the spiked concentrations (300 and 600 μM, respectively), and the signal returned back to the baseline (within 2%) when the sensor is returned to melamine-free milk.