Paraytec’s multi award-winning product*, the ActiPix® D100, is the world’s first UV area imaging detector. It utilises Paraytec’s patented ActiPix technology. This brochure describes the innovations at the heart of this technology. It includes an overview of Paraytec’s flagship product, the ActiPix D100, introduces exciting new applications demonstrating the unique advantages of ActiPix technology, and describes its main features and benefits over existing techniques.

Introduction

Paraytec’s ActiPix technology introduces a new dimension to one of the most widely used techniques in analytical chemistry: ultra-violet (UV) absorbance detection. UV detection is used for the characterisation of a diverse range of liquid samples, including pharmaceutical and biopharmaceutical drugs, peptides, proteins and DNA.

The ActiPix D100 is the world’s first UV area imaging detector. Area imaging is the term which describes the unique capabilities of the 9 mm x 7 mm active pixel sensor array which is at the heart of ActiPix technology used to capture area images in real-time.

ActiPix imaging innovations

Unique self-referencing

When light is shone through a liquid-filled vessel, such as a glass capillary, the liquid inside the capillary and the capillary

vessel combine to act as a cylindrical lens, as shown in Figure 1.

The white band surrounded by an area of shadow is the image obtained of the focused light. When no liquid is present, as

demonstrated by the presence of an air bubble located in the middle of the capillary, there is no focusing effect. When this vessel is placed above an area imaging array, the light in the centre of the capillary is readily referenced against light from the same source using pixels in the dimension transverse to the capillary. An active pixel sensor array is used in the ActiPix imager. This patented ‘self-referencing’ is what makes ActiPix technology unique. It can be illustrated by means of a ray tracing diagram shown in Figure 2.

Figure 2 : Ray diagram through capillary illustrating the patented self-referencing system used in the ActiPix

The light focused through the centre of the capillary is shown in blue, the reference light is represented by yellow, and the boundary between reference and sample is represented by the purple lines. The major benefit of using this self-referencing process is that it is independent of any light fluctuations, resulting in a very stable absorbance signal output. The same process is also visualised in rectangular channels, e.g. in rectangular cells or with channels in lab-on-a-chip devices.

Absorbance, A, is given as A=log Io/I where Io and I are intensity of the incident and transmitted light respectively. To measure an absorbance change of 20 µAU (i.e. 20x10-6) fluctuations in Io and I have to be correlated with a precision of 40 ppm or better. Use of an area imager offers a great advance over existing technologies where two separate sensors are normally used to monitor sample and reference beams. This feature enables use of a wide range of light sources ranging from deuterium to pulsed xenon flash lamps.

The world’s first UV area imaging detector

Row of pixels

Figure 1: The self-focusing effect of shining light through

a liquid-filled tube

High speed area imaging

The active pixel sensor used in ActiPix technology has very high spatial resolution (7 µm). A benefit over charge coupled devices (CCDs) is that the processing and control functions are smaller and consume less power. The instrument design allows high speed detection across the entire imager area in real time with good signal-to-noise and a wide dynamic range.

This technology has been applied to monitor processes in multiple capillaries in an array and opens up exciting new possibilities for multiplexing never before possible. Each capillary acts as a combined UV lens and sample vessel; no external optics or expensive UV lenses are required.

Optimised light transfer

In absorbance detection, the signal-to-noise ratio scales with the square root of the light intensity. In conventional UV detectors which are used for analysing samples passing through a capillary, the diameter of the light source, e.g. a deuterium or pulsed xenon discharge, is typically ~500 µm. The internal diameter of the capillary is typically 50 -100 µm. Therefore, a significant amount of light is lost as it is not possible to create an image brighter than the source. In the ActiPix D100, the process of light transfer through the capillary is optimised by the use of a round-to-line fibre optic cable. The line end of the cable provides an illumination source 100 µm wide and 9 mm long, which for illumination of a long section of capillary provides a significant improvement over conventional light sources. This is shown schematically in Figure 3.

High sensitivity without sacrificing spatial resolution

A final but significant feature of ActiPix technology is in real time signal processing algorithms. As an analyte travels through the capillary, a series of individual snapshots are

Figure 3 : Use of round-to-line fibre optic cable to optimise light transfer from source to capillary

* PittCon 2007 Editors’ Choice Silver AwardR&D 100 Award (2007)Eurolab 2008 Best Trade Offer

taken using the imaging array. The pulsed xenon lamp may be operated at up to 60 Hz. Up to 300 snapshots can be taken in the 5 seconds it typically takes an analyte to traverse the array. The signal processing algorithm determines the velocity and uses an appropriate time displacement to add together and average the snapshots. The analyte signals are correlated whereas the noise is uncorrelated; the net result is a gain in signal-to-noise ratio proportional to the square root of the number of snapshots. This process is shown in Figure 4. In other words, ActiPix technology enables increased sensitivity without sacrificing spatial resolution. The spatial resolution in the capillary direction is 70µm, obtained by combining ten 7 µm imager pixels (a process known as ‘binning’). In a standard capillary electrophoresis system, the width of the window used with single point detection is an order of magnitude greater and the spatial resolution thus an order of magnitude less. The spatial resolution in the direction transverse to the capillary is 7 µm, allowing clear distinction between signal and reference pixels.

conventional

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window, 50 µm ID

D2 or Xe discharge

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Figure 4 : Signal processing via addition of time displaced snapshots

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The world’s first UV area imaging detector

In line quantification and sizing of biopharmaceuticals

BSA at 1 mg mL-1

Hydrodynamic radius 3.35 ± 0.05 nm

Aggregation (the tendency of similar or related species such as proteins to form dimers, trimers or aggregates of the same species) is a major problem during biopharmaceutical manufacturing. The ActiPix can be used to monitor this process quickly and effectively when coupled to a capillary column which has been used to separate these species by CE or nanoLC.

UV used for both quantification and sizing – two key measurements with a single instrument, unlike light scattering techniques that provide just size measurement

Readily coupled to separation stage – quantify and size all molecules in mixtures

High dynamic range: nM – mM

Uniquely applicable to all size species ranging from small (e.g. drug candidates) to large (e.g. proteins and aggregates)

Applicable to proteins in biological media, unlike SDS PAGE gels

Delivering real application advancesThe ActiPix UV area imager offers a powerful new tool for monitoring physical processes in real time.

In this simulation a membrane is placed in the centre of the imaging zone and the migration of UV active species, such as drug candidates, chloride or potassium ions, is monitored. In this mode the ActiPix offers a powerful new tool for drug candidate evaluation, blood brain barrier and active and passive transport studies.

Real-time imaging of transport from one region to another across boundary

Benefits applicable to any species with UV absorbance, unlike fluorescence where labelling is required

Allows key species e.g. K+, Ca2+, peptides to be probed directly

Diffusion across a membrane

Transport and diffusion measurements

membrane

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Size determination by imaging band broadening in pressure-driven flow through a capillary loop

This application shows the ActiPix UV area imager being used to monitor the conversion of glutathione to its oxidised form as it is electrically driven through a zone of hydrogen peroxide.

ActiPix offers combined reaction and separation – applicable to many biological assays

Detect separated species prior to and after passage through a microreactor

Widely applicable to flow chemistry

Broad applicability e.g. enzyme, substrate or inhibitor screening

Real-time imaging of reactions

To view the latest application advances with the ActiPix D100 please visit www.paraytec.com/applications

Lab-on-a-chip UV area detection

Using the ActiPix, separations and other processes in channels can be readily visualised in real time, laying the foundations for applying UV imaging to lab-on-a-chip devices.

ActiPix offers ready integration with lab-on-a-chip devices

Enables methods in the UV down to 190 nm with high sensitivity and high spatial resolution

Visualisation across a 9 mm x 7 mm area detector – nothing else can do this

Electropherogram of p-nitrophenol in microfabricated separation device

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Sample: 24 fmol p-nitrophenol

Buffer: 50 mM phosphate, pH 7.5

Separation voltage: 1000 V

Five snapshot images taken at 1 second intervals showing the injection of p-nitrophenol into a microfabricated device

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Paraytec Limited

1a St Georges PlaceTadcaster RoadYorkYO24 1GNUK

Phone +44 (0)1904 526270Fax +44 (0)1904 652101

Email info@paraytec.com

Web www.paraytec.com

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Instrument Specifications

Light source Pulsed xenon lamp

Fibre optic cable Round-to-line (1 m total length)

Wavelengths 190 – 1200 nm, filter selectable

Filter bandpass 10 nm

Peptide limit of quantification 100 ng mL-1

Baseline noise 5 µAU RMS (1 second time constant)

Linear dynamic range 5 orders (0.005 mAU – 500 mAU)

Spatial resolution 70 µm

Pixel size 7 µm x 7 µm

Imager area 9 x 7 mm (L x W)

Sensor head size 6 x 6.5 x 11 cm (H x W x L)

Control box size 14 x 22 x 25 cm (H x W x L)

Total system weight 7.5 kg

Power 100 – 240 V AC 50/60 Hz

Environment 10 – 30°C

Safety Certified to UL61010-1:2004IEC61010-1:2001