Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
2002-12-11
2004-11-02
Barlow, John (Department: 2863)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C073S864840, C422S091000, C435S286500
Reexamination Certificate
active
06813568
ABSTRACT:
BACKGROUND
This invention relates to the field of microfluidics-based automated chemistry.
Many analytical chemical problems involve the analysis of small amounts of a sample. The ability to do chemical reactions in small volumes of liquid is important in such cases as the reaction rate can decrease to commercially unacceptable levels if the concentration of the sample is too low. Additionally, for analytical devices used to monitor the results of such reactions, it is frequently important that the reaction product be in a small volume in order that a detectable concentration be present. The present invention offers a solution to the problem of doing chemical reactions in small volumes, to doing a succession of such reactions, and to doing them on an automated basis.
Although the present invention will be seen to have general chemical applicability, its application to biomedical research is of particular interest. Post-genome research will likely focus on understanding the cellular chemistry, circuitry and communications underlying life's vital processes. Biomedical scientists, for instance, will aim to identify how genetic determinants of disease alter cellular physiology and response to agonists. Predictably, all this will involve biochemical analysis of larger numbers of samples containing ever lower concentrations of analyte. In most cases, analyses entail multistep procedures, including chemical and enzymatic reactions. Not only will automation prove essential in such cases, it must also be done in fully integrated instruments that incorporate the smallest possible reaction vessels and wetted surfaces. Progress towards this goal has come from nano-fabricated devices
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(“lab-on-a-chip”), but severe limitations on sample volume may restrict the technology to fast, parallel analysis of abundant and/or amplifiable (e.g. by PCR) molecules. What's needed is automation whereby trace analytes are processed in their entirety. We wanted to construct such a device, initially using chemical protein sequencing as a model system. Such chemistries have been replaced during recent years by mass spectrometry as a means to protein identification.
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Yet, the fact remains that chemical analysis can yield rather long stretches of easy to interpret sequence, including on intact proteins, with the caveat that current instruments are at least ten to twenty times less sensitive than most mass analyzers.
Traditionally, protein chemical sequencing is done by stepwise removal of amino acids from the N-teminal end, one at a time. In this method, the Edman degradation,
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phenyl isotliocyanate (PITC) is coupled to the alpha-amino group of the polypeptide to form a phenyl thiocarbamyl (PTC)-derivative; anhydrous acid causes selective release of the PTC-amino, leaving a truncated peptide chain. The resulting anilino thiazolinone amino acid (ATZ-aa) is converted with aqueous acid to a more stable phenyl thiohydantoin amino acid (PTH-aa) and identified. The latter step cannot be done in the presence of polypeptide as it would cause hydrolysis; thus, the ATZ-aa must be extracted. The procedure was partially automated, first by Edman in the ‘spinning cup’ version,
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and later in Laursen's solid-phase sequencer.
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Conversion was initially done outside the machine, but later incorporated into the automated process by Wittman-Liebold.
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Until then, thin layer or gas chromatography were used for PTH-aa identification; Hunkapiller and Hood were among the first to routinely use reversed-phase high performance liquid chromatography (RP-HPLC) for this purpose.
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Around 1980, the gas-phase (GP) sequencer was developed by Hewick and coworkers.
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Here, wash-out of the peptide from the reaction vessel was prevented by non-covalent immobilization on a glass-fibre disc and by delivering polar liquids as vapors. The process has been further automated by coupling HPLC identification of PTH-aa's ‘on-line’ with the sequencer; contents of the conversion flask are hereby directly transferred into the LC injector loop. Since then, progress has come from incremental improvements such as femtomole level HPLC detection,
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rigorous instrument optimization and maintenance routines,
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and the use of a smaller reaction cartridges.
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Combined with improved micro-preparation of polypeptides,
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chemical sequencing at the 1-3 picomole level is currently possible and extended sequencing runs with femtomole level signal have been reported.
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Additional modifications to the process have since been suggested that should allow true femtomole level sequencing. All relate to increased sensitivities of amino acid derivative detection. This can be accomplished by miniaturizing the HPLC-based PTH-aa detection system, or by producing modified Edman end-products of higher detect ability, or both.
Whereas femtomole level PTH-aa separations on microbore columns can be done,
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the use of capillary columns (300-micron ID) is more problematic, as injection volumes of over 0.5 &mgr;L cause baseline disturbances.
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Complete injection (or even 10%) of the sample in a 0.5-&mgr;L volume is not possible from any commercial automated sequencer. This also applies to capillary zone electrophoresis (CZE)-based amino acid-derivative detection systems.
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Considerable effort has been expended to generate fluorescent amino acid derivatives as end-products of Edman chemistry. Fluorescent sequencing is especially appealing since the introduction of sub-attomole amino acid analysis by CZE with laser-induced fluorescence.
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Yet, again, loading volumes are in the nanoliter range, several orders of magnitude less than what is typically present in a sequencer flask. Another approach to ‘high-detect ability’ is the generation of quaternary or tertiary amine thiohydantoin amino acid derivatives, analyzable by mass spectrometry at the low femtomole level.
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However, both methods never progressed beyond the R&D stage.
Development of any Edman based, femtomole-level technique requires satisfying two major criteria: (i) quantitative transfer, in the smallest possible volume, of amino acid derivatives to the site of analysis; and (ii) reducing chemical background that will impede any ultra-sensitive analytical technique. This can only be accomplished by further miniaturization of the chemistry.
We describe a microfluidics-based instrument, consisting of multiple rotary valves, capillary tubing and miniaturized reaction vessels, for the purpose of performing automated chemical and biochemical reactions on a very small scale. Close to 100% of the reaction end-products are available in a minimal volume (≦5 &mgr;L) inside a pressurized mirco-vial for subsequent analysis. This makes the system compatible with capillary HPLC and, in principle, with continuous flow nano-electrospray mass spectrometry. Total control of flow path combinations and directions, temperatures and gas pressures enables precise execution of complex biochemical laboratory procedures. Instrument performance was convincingly demonstrated by partially sequencing 100 femtomoles of an intact protein using classical Edman chemistry in combination with capillary-bore liquid chromatography. To our knowledge, this is the smallest amount of protein ever reported to be successfully analyzed in this way.
SUMMARY OF THE INVENTION
The invention is a chemical system and related processes that utilize small-volume rotary selector valves and small-volume rotary switching valves in combination under the control of a computer. The small rotary valves are particular well-suited to computer control. As a result, a single system can be programmed to do a variety of tasks by changing the program but leaving the system's components substantially intact.
As a result, in a general aspect the invention is a system for carrying out one or more chemical reactions, said system comprising a rotary selector valve and a rotary switching valve, each valve under the control of a computer, the internal volumes of the selector and switching valves each being 1.5 &m
Powell Michael
Tempst Paul
Barlow John
Klauber & Jackson
Le John
Memorial Sloan-Kettering Cancer Center
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