Overcoming challenges encountered by PCR filters in high-performance qPCR

Engineers developing polymerase chain reaction (PCR) instrumentation are faced with extraordinary issues related to both the quantitative detection of nucleic acid sequences using real-time analysis and the qualitative detection of nucleic acid sequences (using end-point analysis).

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Quantitative PCR (qPCR) instruments that function in real-time require an optimal signal-to-noise ratio, in combination with the utmost sensitivity.

Due to this kind of demand for high sensitivity, the bandwidth of excitation and emission bandpass filters and center wavelength (CWL) utilized for the configuration of each channel of a qPCR instrument must be optimized based on that instrument’s system configuration, as well as the number of channels it needs to support.

The CWL and bandwidth for each filter must maximize both the emission and excitation signals while reducing cross-talk between signals fixed into the same channel and eliminating interference with adjacent signals. 

The sum of its parts

Just as each qPCR instrument is the sum of its channels working in harmony with one another, the optical filters directing each channel are best thought of as a group rather than separate components.

Similar to other fluorescence applications, the emission bandpass filter, the excitation bandpass filter and the dichroic filter on each channel carry out specific roles that are interconnected. For instance, the excitation filter sends the desired illumination wavelengths and impedes any unwanted signals.

The emission filter only allows specific wavelengths related to the desired fluorophore through to the detector, and the dichroic filter is applied to guide the various excitation and emission wavelength ranges to different beam paths. Each filter’s performance affects and is affected by its associates.

As with a number of similar applications, the quality qPCR instrumentation is based on the components that optimize performance at an accessible price point. As each channel on a qPCR instrument is similar to its neighbors in wavelength, technological challenges occur, especially when more channels are added.

Costs to mitigate these issues can rapidly grow out of hand — especially when considering most bandpass and dichroic filters for these instruments necessitate a custom design, integrating certain fluorochromes or wavelengths.

Interference with the detection of the emission signal can be caused by an overlap between the excitation signal and the emission signal. Thus, it is absolutely crucial to employ bandpass filters operating with an extremely steep edge, thus producing improved signal isolation due to high transmission on the passband and deep blocking outside the passband.

Standard, commercial off-the-shelf filters may be suitable in some applications, but more than 90% of optical filters in qPCR instruments will necessitate some form of customization to meet their intended price/performance balance.

Therefore, it is crucial to engage early with a vendor to avoid selecting a part that may be inappropriate for the project or miscalculating a part’s price/performance balance.

Ideally, engaging early with a vendor also grants access to invaluable input before an instrument’s design is completed, resulting in the best technical and commercial optical filter (or series of filters) being used predicated on the customer’s requirement/ instrument configuration.

Generally, wavelength range and CWL make up the most common tweaks applied to qPCR filters. However, transmission level, bandwidth and refraction level remain key parameters to optimize.

How Iridian can help

Instrument makers require robust, reliable solutions that are also cost-effective. However, in reality, there are a number of other challenges such as design and component costs, on-time delivery, quality considerations, and simply finding a high-performance filter partner that works to understand and execute your vision.

The difficulty of each challenge is contingent on the individual project: one instrument maker may have difficulty meeting the technical demands its product presents, while another may struggle to locate a vendor whose supply chain can scale from prototype to volume. 

From a technical standpoint, Iridian ultra-performance filters have been specifically developed for LED-based, real-time PCR applications.

Iridian specializes in working closely with customers to develop and implement customized filter solutions that maximize their instrument’s effectiveness. Its standard filter solutions are compatible with common excitation sources and fluorophores. 

For instance, Iridian’s ultra PCR fluorescence filter series has been developed to offer a high signal-to-noise ratio of the probes in real-time PCR samples when illuminated with an LED light source.

Iridian’s engineers are able to customize distinct spectral characteristics or physical filter sizes. Each of these custom filters — excitation, emission, and dichroic — offers more signal, with less background, to both fluorescence instrument users and OEMs.

They offer greater transmission within the passband and deep blocking beyond the range of the passband: Iridian’s excitation and emission filters incorporate steep edge transition (<0.5% from 50% transmission to blocking OD5), whereas the company’s dichroic filter features < 1% slope from 10% to 90% transmission point. 

Perhaps most importantly, Iridian PCR filter solutions can guide a project from “good idea” to “good product.” These filters can be scaled from early prototype through to high-volume manufacture without compromising on the final quality. 

Certified to ISO 9001-2015, Iridian’s facilities and processes also stand up to the scrutiny of the industry’s regulatory guidelines. Iridian operates a stable, high-yield, and high-volume filter production process, guaranteeing a supply chain its customers can trust.

Furthermore, Iridian’s scrupulous testing and process control offer consistent filter performance from filter-to-filter and batch-to-batch, plus complete traceability for all of its products.

Conclusions

Iridian Spectral Technologies has expertise across a diverse range of applications, spanning fiber optic communications and optical spectroscopy to 3D entertainment and aerospace instrumentation. This vast experience offers insight into the regulatory, technical, and logistical challenges that Iridian customers face on a daily basis.

Nonetheless, qPCR instrument-related challenges can be among the most intimidating, weighed down by tough deadlines, tight budgets, and expectations for immaculate functionality.

A combination of Iridian’s excitation, emission, and dichroic filters serving each channel of a qPCR instrument allays a number of these concerns, offering the greatest sensitivity while maintaining a desirable signal-to-noise ratio.

For more information, visit: www.iridian.ca.

About Iridian Spectral Technologies Ltd.

Iridian Spectral Technologies Ltd. has maintained itself as a world leader in custom optical filter solutions since its founding in 1998 and we pride ourselves on supporting our customers optical products across a wide range of geographic and market areas. We have at our core the belief that if our customers succeed we succeed as well.

Our team at Iridan uses decades of filter design and manufacturing experience to provide support throughout a product’s life cycle; from initial prototyping to high volume manufacturing and our automated production facility is able to ensure that the most cost effective optical solutions are found for any application.

Iridian’s highly advanced, proprietary thin-film design deposition and manufacturing technology delivers durable high performance filter optics for use in telecommunications and data centers, Raman and fluorescence spectroscopy, mid IR applications such as gas sensors, as well as astronomy and aerospace.


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Last updated: Jun 15, 2022 at 8:59 AM

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