Overcoming challenges faced by PCR filters in high-performance qPCR

Engineers developing polymerase chain reaction (PCR) instruments face extraordinary problems related to both quantitative detection of nucleic acid sequences using real-time analysis and qualitative detection nucleic acid sequences (using endpoint analysis).

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Quantitative PCR (qPCR) instruments that operate in real time require an optimal signal-to-noise ratio, combined with maximum sensitivity.

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

The CWL and bandwidth of each filter should maximize both transmit and excitation signals while reducing crosstalk between fixed signals in the same channel and eliminating interference with adjacent signals.

The sum of its parts

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

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

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

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

The costs of mitigating these issues can quickly spiral out of control, especially considering that most bandpass and dichroic filters in these instruments require custom design, incorporating certain fluorochromes or wavelengths.

Interference with transmit signal detection may be caused by an overlap between the excitation signal and the transmit signal. Thus, it is absolutely crucial to use bandpass filters operating with an extremely steep edge, thus producing better signal isolation through high transmission on the passband and deep blocking outside the passband.

Off-the-shelf filters may be suitable for some applications, but over 90% of qPCR instrument optical filters will require some form of customization to achieve the intended price/performance balance.

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

Ideally, engaging early with a supplier also provides access to invaluable information before an instrument design is complete, allowing the best technical and commercial optical filter (or series of filters) to be used in depending on customer requirements/instrument configuration.

Typically, wavelength range and CWL are the most common settings applied to qPCR filters. However, the level of transmission, the bandwidth and the level of refraction remain key parameters to be optimized.

How Iridian can help you

Instrument manufacturers need rugged, 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 depends on the individual project: one instrument manufacturer may struggle to meet the technical requirements of their product, while another may struggle to find a supplier whose supply chain can scale from volume prototype.

From a technical point of view, Iridian high performance filters have been specifically developed for LED-based real-time PCR applications.

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

For example, Iridian ultra PCR fluorescence filter series was developed to deliver high signal-to-noise ratio of probes in real-time PCR samples when illuminated with an LED light source.

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

They offer better transmission in the passband and deep blocking beyond the passband range: Iridian’s excitation and emission filters incorporate a steep edge transition (<0.5% of the 50% transmission at OD5 blocking), while the company's dichroic filter has a <1% slope from 10% to 90% transmission point.

Perhaps most importantly, Iridian PCR filter solutions can take a project from “good idea” to “good product”. These filters can be scaled from first prototype to high volume manufacturing without compromising final quality.

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

In addition, Iridian’s scrupulous process control and testing provides consistent filter performance from filter to filter and batch to batch, as well as full traceability for all of its products.

conclusion

Iridian Spectral Technologies has expertise in a wide range of applications, ranging from fiber optic communications and optical spectroscopy to 3D entertainment and aerospace instrumentation. This extensive experience provides insight into the regulatory, technical and logistical challenges that Iridian customers face on a daily basis.

Nonetheless, qPCR instrument challenges can be some of the most daunting, weighed down by tight deadlines, tight budgets, and expectations of flawless functionality.

A combination of Iridian’s excitation, emission, and dichroic filters serving each channel of a qPCR instrument alleviates a number of these issues, providing 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 the global leader in custom optical filter solutions since its inception in 1998 and we are proud to support our customers’ optical products in a wide range of geographies and markets. We believe that if our customers succeed, we also succeed.

Our team at Iridan uses decades of experience in filter design and manufacturing 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 proprietary, highly advanced thin-film deposition and fabrication technology provides durable, high-performance optical filters for use in telecommunications and data centers, Raman and fluorescence spectroscopy, mid-infrared applications such as gas sensors, astronomy and aerospace.


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