Principles of Sequential
and Bead Injection

Injection of a well defined volume of analyte ( red zone) into a carrier stream of a buffer is the first step (A) of the experimental protocol.  Next (B) a precisely metered volume of reagent (dark blue) is injected, pushing the analyte zone upstream into the holding coil. As these sequentially injected zones mix on contact (C)  a product (yellow) begins to form. Then the flow is reversed (D) to promote further mixing and to transport the composite zone into a flow through detector. For very fast reactions the reaction nears completion and a peak is recorded, while  the fully developed product is passing  through the  flow through detector  (E).  The response curves obtained by Sequential Injection when analyte zone is flowing through the detector are similar to those obtained with traditional continuous flow injection.  Since typically only a few seconds are needed to complete sequences A-D, a longer incubation time is often needed to generate a measurable amount of the reaction product. By stopping the flow, while the composite zone is within the flow through detector  reaction rate is measured in real time – an approach that provides the essential information for accurate assay. Kinetic assay of glucose, showing five reaction rate curves (superimposed using graphics of  FIAlab software),  is example of this approach which eliminates the influence of blank value. 

To summarize, in  contrast to classical Flow Injection, or air segmented flow based assays,  that operate in the continuous flow mode, Sequential Injection is based on forward-reversed-stopped flow. This microfluidic programming  promotes mixing and saves reagents since  the liquids are flowing intermittently and only when the sample is being processed.

Yet another  advantage of Sequential Injection is that that all parameters of the experimental protocol: sample volume, sample dilution, reagent volumes, mixing and incubation time are selected  solely through software control, by adjusting  the stroke and direction of the pump and selecting appropriate port of the multiposition valve, thus  achieving  a  random access to sample, standards and reagents in any  desired sequence. Thus while flow channels of conventional flow analyzers must be  reconfigured when changing from one type of assay to another one, LAB-ON-VALVE system will  accommodate various experimental protocols by changes of software controlled microfluidic programming (LOV and Sequential Injection).
 

Bead Injection operates in the same fashion as Sequential Injection, albeit  using microspheres as reagent carriers. Injection of a well defined volume of beads (A) is followed by injection of a analyte zone (red). The beads are captured within the flow trough detector, where their optical properties are monitored. As the sample zone arrives, the analyte reacts with the functional groups on bead surfaces, changing their optical properties and  yielding a response curve that is the basis of a given assay (B,C). Upon completion  of measurement the beads are discarded (D).

The capability of   Bead Injection technique applied to is shown here on two examples of  Bioligand Interaction Assay. Sepharose 4B   Protein G coated beads were captured in a flow cell and then perfused by IgG followed by PBS buffer wash, while the absorbance of the bead layer was monitored at 262 nm. The recorded curves show increasing accumulation of IgG with increasing amount of injected antibody and stability of the formed biocomplex which does not dissociate during the wash phase. The response curves, obtained in separate experiments, each on fresh portion of beads ( 2 microliters ) are shown superimposed. Use of UV-VIS Spectroscopy for monitoring of bioligand interaction allows simultaneous  monitoring of unlabelled as well as labeled biomolecules – such as monitoring of FITC labeled IgG whre protein was monitored at 273nm and FITC at 476 and 494nm.  UV-VIS monitoring is a  unique advantage of BI   compared to traditional systems ( such as BIAcore) that use surface plasmon resonance as detector.

Bead Injection allows selective accumulation of target molecules at renewable and disposable surface. These  interactions can be either monitored in real time, or the beads can be further processed by elution ( for MS)  or by ashing ( for ETAAS). This variant of BI  has been termed as  renewable microcolumn technique ( LOV and Bead Injection ).

Flow Injection, Sequential Injection and Bead Injection are related, mainstream techniques for reagent based automated assays. While Flow Injection has been around for 25 years, resulting in publication of over 11.000 papers and numerous monographs ( Literature), Sequential and Bead Injection are more recent, fully computerized techniques,  brought to ultimate microminiaturization within  the LAB-ON-VALVE module. The numerous assays developed for Flow Injection format can be easily adapted to LAB-ON-VALVE system.