The development of microfluidics for clinical diagnostic use opened a new chapter in the accessibility of lab tests known as “lab-on-a-chip.” These devices bring robustness, reproducibility, and—in particular—miniaturization that is hard to achieve with earlier technologies. From the start, microfluidic technology has depended on plastic for its primary components: plastics can form complex shapes with precision at a microscopic scale and be manufactured in bulk at low cost compared with other materials.

Yet plastics are prone to autofluorescence that can interfere with the performance of a diagnostic device. Many materials autofluoresce at a level that isn’t noticeable to the human eye. However, if you include these materials in a diagnostic device intended to detect or measure fluorescent signal, there can be significant issues.

What is Autofluorescence?

Autofluorescence is the natural emission of light by a material when it has absorbed light. Different materials have different degrees of autofluorescence effects. Some plastics, especially transparent plastics such as COC, COP, and PMMA, tend to have more autofluorescence than glass.

Why is Autofluorescence Important in Device Development, Manufacture, and Use?

Autofluorescence acts as a background noise in diagnostic applications. It can interfere with the reading accuracy of fluorescent signals. This includes detection of reagent mixing, monitoring or reaction progress, and recognition of washing efficiency to achieve consistent reagent concentrations. At worst, it can also obscure final test results of diagnostic applications if directly dependent on the fluorescent signals.

In some cases, you may have finalized your device design (or even created initial prototypes) and are now prepared to transition to hard plastics for injection molding. Special considerations related to autofluorescence are essential during your transition to a new material, particularly when optical monitoring or detection are involved in the operation of the chip.

Can You Eliminate Autofluorescence from Transparent Plastic Microfluidic Chips?

The amount of autofluorescence varies among the plastics used in device manufacturing and varies even more depending on the wavelength of light that excites it. In addition, testing the raw materials before prototyping isn’t sufficient—studies have found that plastic microfluidic chips will have more autofluorescence than their material did before processing. While it isn’t possible to completely avoid autofluorescence, you can minimize its impact. This requires proper knowledge of material grade, channel designs, and injection-molding methods that lessen the overall effect and avoid disruptive interference.

How to Mitigate Autofluorescence Effects on Plastic Microfluidics

During Product Development

It is essential to integrate autofluorescence considerations with your overall product development process. Here are the key steps:

Measure the autofluorescence level of your product and analyze its cause
Determine the optimal material grade and channel design to reduce the amount of autofluorescence.

Measurement and Analysis

To develop a device with minimum autofluorescence interference, you must begin by measuring the amount of fluorescence in conditions that match those of the chip’s intended use. Understanding the range of end-user settings is crucial: physician office labs and clinical diagnostics labs may have different environmental controls, lighting, or even storage conditions of the chips and reagents. Some devices may be intended as manufacturing controls in a factory setting, bringing additional variables to the fore. As part of your design control process, take stock of the full range of conditions across these settings, including humidity, temperature, consistency of reagent use, reagent concentration, chip and reagent storage conditions, light sources, the light’s excitation wavelength and excitation filter, and others that you uncover during your end-user characterization process. Depending on the application or the device, a specific fluorescence, including FAM, HEX, GFP, or Cy5, may need to be measured.

Expert Support for Autofluorescence Evaluation

Enplas can help you define and execute your evaluation of your prototype’s autofluorescence challenges. Our customizable in-house evaluation equipment can replicate the environments in which your customers will use the device and measure autofluorescence levels in those conditions. For Enplas to assist with this measurement, you must provide the reagent itself (or information about which reagent needs to be used). Our evaluation equipment includes the light sources and filters necessary to measure a range of fluorescence including FAM, HEX, GFP, and Cy5. We can support autofluorescence evaluation for any optical detection applications.

Figure 1 shows how the right measurement process can identify areas within your channel design that affect excitation and emission of fluorescence during product use.

Figure 1: Measuring Autofluorescence in Intricate Channels

In addition to conducting standard measurements, Enplas can measure autofluorescence in harder-than-normal situations. For example, Enplas can measure fluorescence from the side of the channel (as shown in Fig. 1). This is more difficult to execute than the more common approach of measuring from above, but it enables measuring autofluorescence in thin wall channels.

Material Grade and Channel Design

The intensity of autofluorescence varies with material grade. Choosing the right material grade of COC, COP, or PMMA can lower the barriers to your successful management of signal interference. Thoughtful channel design informed by the thorough measurement and analysis process described above can significantly reduce autofluorescence. There are several techniques to consider:

Thinning the chip thickness at the detection area could minimize autofluorescence for in-channel fluorescent detection.
Secondly, increasing the contrast between target detection fluorescence and the background fluorescence can also eliminate noise. Target fluorescence can be intensified by deepening the channel depth.
Finally, reducing the thickness of material used for capping the microfluidic channels is very important: films are the most effective. In general, thin films tend to sag and cause issues, but Enplas has lamination technology to bond thin films onto microfluidics without sagging even when the coverage area is a wide channel or large chamber.

During the Product Manufacturing Phase

As the manufacturing phase begins, your design control principles continue to apply. Manufacturing your product with precision based on observations during product development is crucial for mitigation of autofluorescence on plastic microfluidic chips. The manufacturer must be knowledgeable about selecting and preparing the right cleanroom environment, equipment, and machine condition. Design alone cannot prevent autofluorescence. Microfluidic chips which have been flawlessly designed to reduce autofluorescence can be ruined if the right injection molding, monitoring, and maintenance are not conducted.

Molding Process

Autofluorescence effects tend to be stronger when plastics are exposed to higher temperatures for a longer time. However, there are times when high temperatures or long cycle times are unavoidable (e.g., in microfluidics with intricate designs). Scientific molding and microfluidics molding expertise is crucial to identify the ideal molding process to ensure uninterrupted manufacturing of quality chips.

Injection Molding Machine Condition

In precision manufacturing, the condition of the specialized equipment cannot be taken for granted. The smallest particle that might be unimportant in standard injection molding of products can be a real problem when the goal is to reduce autofluorescence effects relating to sensitive microfluidic chips. Meticulous preparation for each manufacturing batch is essential, including:

Setting the right amount of heating time
Creating the proper cleanroom environment
Conducting the appropriate purging
Frequent overhauling of each machine

The above are important processes for creating optimal conditions.

Expertise and Proprietary Methods

Enplas has developed a proprietary manufacturing process that optimizes autofluorescence mitigation. Enplas may also designate an injection-molding machine to exclusively run projects that use the same materials as the product requiring low autofluorescence. This eliminates any residual miniscule materials that may negatively affect autofluorescence for applications with strict requirements. The presses are monitored in real time to ensure our autofluorescence-mitigating molding process is on track. We offer ISO Class 6 to Class 8 cleanrooms, depending on your company’s need.

In Figure 2 below, each group of plots represents one production day and shows the measurement of autofluorescence intensity of microfluidic chips injection-molded during that day. (Blue plots represent Day 1, and orange plots represent Day 2.) The data show how unregulated injection molding (Day 1) can yield chips with high fluorescence intensity despite good product design. On Day 2, Enplas improved the injection-molding process so that all shots stayed in the low fluorescence range (orange plots).

Figure 2: Benefits of an Optimized Manufacturing Process for Reduction of Autofluorescence

Generally, as the injection molding makes more shots, the level of autofluorescence decreases (Fig. 2). However, there are times when unexpected issues cause a sudden increase in autofluorescence. To prevent this, Enplas monitors the process throughout the entire running time. If any abnormalities are detected that cause high fluorescence, the run can be stopped and the process can be adjusted to prevent loss of time or product.

Conclusion

Autofluorescence of plastic microfluidic chips is inevitable. For any product that involves accurate reading of fluorescence, it must be considered in the design control process to achieve its desired result. Autofluorescence effects on plastic microfluidic chips can be reduced with proper decision and handling during the design development and manufacturing phases. It is important to work with a manufacturer that has the expertise and technology to help you produce microfluidic chips with minimized autofluorescence.

Enplas conducts original research to study autofluorescence effects on plastic microfluidic chips and applies that expertise to help customers during the design development phase. Enplas supports you in achieving optimal product design with low autofluorescence interference. The microfluidic chips mass produced by Enplas will be delivered with stable levels of reduced autofluorescence.

Contact Enplas today for consultation on how you can mitigate autofluorescence on your plastic microfluidic device.

DID YOU KNOW?

Enplas can manufacture plastic microfluidic chips in the millions/year with consistent high quality! Quality assurance systems highly tailored to microfluidics manufacturing and cutting-edge equipment make this possible. Contact us to find out more!