DE102012013241A1 - Microfluidic flow cell for use in arrangement for cavity ring-down absorption spectroscopy for detecting smallest analyte concentration, has optically transparent wafer that is arranged in flow measuring chambers - Google Patents

Abstract

The microfluidic flow cell (1) has an optically transparent wafer (2) that is arranged in flow measuring chambers (21,22) with inflow and outflow channels (21a, 21b,22a,22b). The intensity of light beam (L) irradiated perpendicularly with respect to flow measuring chambers is three times the light beam diameter (dL). The optically transparent plates (11) are connected to flow measuring chambers. The chamber surface opposite to light beam area is provided with immobilized nano particles, and the height (hg) of wafer parallel to light beam direction is set below 1 mm.

Description

The invention relates to a microfluidic flow cell for use in a cavity ring-down (CRD) absorption spectroscopy system for detecting lowest analyte concentrations.

Proven feature sizes of microfluidic chips [e.g. B. Mark, et al .: Chem. Soc. Rev., 39, 1153-1182, (2010) ], the z. B. in lab-on-chip systems or other microfluidic platforms use and are usually applied in the flow, are usually in the sub-mm range, ie about 130 microns measuring channel cross-sections (width x height), where there are drops in the channels uniform cross section are examined. The microfluidic chips produced according to this prior art are prestructured, inter alia, by means of known microsystem techniques into two half-chases, which are usually by means of bonding methods (eg anodic bonding [eg. Kutchoukov, et al .: Sensors and Actuators A, 114, 1521-527, (2004) ]) are permanently connected to each other.

The use of plasmonic activities on chemically synthesized metallic nanoparticles is also known as such, but not in the solution form of the invention set out below. Optical evaluations of plasmonically active material are generally carried out in commercially available spectrometer cuvettes (no flow cuvettes) in a liquid environment by means of UV / VIS spectrometry. This approach requires a correspondingly high concentration of nanoparticles and analytes in order to even produce a measuring effect. In this technology, analyte molecules from a liquid selectively bind to the surface of metallic nanoparticles. The plasmonic active nanoparticles (nanosensors) react after binding of the analyte substance with a shift of their resonance frequency due to the changed environmental conditions (LSPR-Localized Surface Plasmon Resonance effect). This is expressed in a small change in absorption or a spectrally altered loss. Spectrally, either a change in the peak height or a shift in the absorption spectrum of the nanoparticles can be observed [ Tapan K. Sau, et al .: Advanced Materials 22, 16, 1805-1825, (2010) ]. The detection in a prior art two-beam spectrometer is only possible if this absorption change is at least 0.001 (1 per thousand) or more. An increase of the measurement signal would be possible by these methods only by a higher particle density, but meets their technically possible limits.

It is also known to apply immobilized particles to microscope coverslips or microscope slides on which the analyte can be detected by means of fluorescence microscopy or evanescent field microscopy.

Microfluidic cuvettes enable the measurement of very small volumes ranging from 1 μl to 1 picoliter. However, the miniaturization goes hand in hand with a drastic reduction in the optical path length of 1 cm in conventional spectrometers to path lengths between 10 μm and 500 μm in microfluidic systems. At the same time, this requires a reduction in sensitivity to values between 1/1000 and 1/20 of the sensitivity in conventional systems. The state of the art in the field of development of chip cuvettes is z. In DE 10 2006 035 581 B3 shown comprehensively.

However, the above-mentioned methods and devices fail to detect the lowest analyte concentrations because, as shown above, they come to their measuring limit as a result of opposing requirements.

Also already known is the method of so-called cavity-ring-down (CRD) absorption spectroscopy, which was initially used for the highly accurate determination of gases and their composition [ G. Berden, R. Peeters, G. Meijer; Cavity ring-down spectroscopy; Int. Rev. Phys. Chem. 19, 565-607 (2000) ]. The investigation of the spectrally resolved reflectivity on highly reflecting laser mirrors by means of a CRD arrangement is described in G. Schmidl, W. Paa, W. Triebel, St. Schippel, H. Heyer; Appl. Optics, Vol. 48, No 35; (2009) described in more detail. In further sources [cf. G. Berden, R. Engeln: Cavity ring down spectroscopy - Techniques and Applications, Wiley 2009 . ISBN: 978-1-4051-7688-0 / Hans-Peter Loock: Trends in Analytical Chemistry, Vol. 7, 2006 / Van der Sneppen, F. Ariese, C. Gooijer, W. Ubachs: Annu. Rev. Anal. Chem. 2009 2: 13-35 ] describes the theoretical basics of the cavity-ring-down method, various fields of application and various cavity configurations. So z. In G. Berden, R. Engeln: Cavity ring down spectroscopy - Techniques and Applications, Wiley 2009 . ISBN: 978-1-4051-7688-0 , set out some applications that use standard flow cells in mirror resonators or microchannels in conjunction with fiber-loop CRD. The use of fiber-loops CRDS (CRDS: cavity-ring-down spectroscopy) with its advantages and disadvantages (low sensitivity) for liquid phase detection in microcapillaries is described in [ Hans-Peter Bock: Trends in Analytical Chemistry, Vol. 7, 2006 ]. Principles and techniques of CRDS or EW-evanscent wave (EWS) on liquids and thin films using cuvettes in mirror resonators or liquids on the surface of a prism resonator are topics, on the in Van der Sneppen, F. Ariese, C. Gooijer, W. Ubachs: Annu. Rev. Anal. Chem. 2009 2: 13-35 will be discussed in more detail.

The present invention has for its object to provide a microfluidic flow cell, which is suitable for use in any arrangement for cavity-ring-down absorption spectroscopy to detect lowest analyte concentrations at low optical path lengths and thus enables the detection of even the lowest analyte concentrations with Previously known methods of the prior art were not detectable. This should also open up the possibility, due to the availability or the price to investigate also analyte substances that are available only in small quantities and therefore could not be examined so far in conventional flow cells.

The object of the present invention is achieved by the features of patent claim 1. Advantageous embodiments are the subject of the dependent claims.

The invention will be explained below with reference to an embodiment. Show it:

1 an advantageous embodiment of the lower part of a microfluidic flow cell according to the invention,

2 a lateral section along a plane XX according to 1 through a complete microfluidic flow cell with separate reference chamber,

3 exemplary arrangement possibilities of a microfluid flow cell according to the invention within differently designed, respectively operated cavity ring-down arrangements based on the examples A to C and

4 an exemplary change in the absorption spectrum when an analyte binds to individual nanoparticles formed according to the invention or a monolayer of nanoparticles.

In order to achieve the object of the present invention, a special flow cell is inventively first created, which is characterized by an extremely small optical path length in the thickness of the measurement path provided. According to the present invention, the flow cell is 1 in a specific example according to the not to scale 1 and 2 initially from an optically transparent wafer 2 , which in the specific example is designed as glass wafers with the external dimensions of 16 x 24.5 mm and a thickness of 700 μm. In this wafer 2 are in the example two chambers 21 and 22 etched, each with also etched inflow and outflow channels 21a . 21b ; 22a . 22b are connected. In the proven and preferred example is the chambers 21 . 22 a size of 3 x 6 x 0.06 mm (b x y x h; 1 and 2 ). How out 2 is apparent, above-mentioned patterned wafer 2 with an optically transparent plate 11 , in particular formed by a 0.2 mm thick quartz plate, covered. On this tile 11 are on the measuring chamber side facing in a previous preparation step at least in one area immobilized individual nanoparticles 12 applied. Called wafer 2 and the slide 11 are by means of a joining substance 13 or an adhesive sheet, or a thermally dissolvable adhesive or adhesive releasable bonding agent, into a reversibly releasable, sealed joint.

Essential in the context of the present invention is the small thickness h _{g of} the entire measuring chamber, which is in the order of less than 1 mm, depending on the thickness of the joining substance, so that an extremely small optical path length when irradiated (see. 2 ). Furthermore, the definition of the dimensioning of the measuring or reference chamber 21 . 22 in the context of the invention essential, because this is to be determined so that the diameter of the waist of the chambers 21 . 22 penetrating light beam L (see that with d _L in 1 designated dotted circle), which designates the diameter at which the intensity of the light beam has fallen to 1 / e, in any case the special design (also other chamber designs with, for example, inflow and outflow channels perpendicular to in 1 , ie in the y-direction, illustrated embodiment are of course possible within the scope of the invention) is not greater than one third of the aperture formed by the chamber boundaries in the smallest extension direction (here x, cf. 1 ). Furthermore, the measuring chamber 21 , and so provided, the reference chamber 22 , Within the scope of the invention, an extremely small height h is given, which in the specific example is h = 60 μm with a width b of approximately 3 mm (cf. 2 ). Basically, the height h of the chambers 21 . 22 be set as low as possible in the context of this invention, depending on the viscosity of the carrier liquid, but so large that a flow through the chamber is ensured with liquid, which is advantageously given in a range of some 10 microns to a few 100 microns, this height h but always very small compared to the lateral chamber expansions (x, y) is selected.

By applying a predetermined selectable coverage of nanoparticles 12 in the microcuvette 1 becomes one of the respective ones Measuring task separately customizable sensor chip formed, which according to the invention can be selectively and sensitively read out further by the cavity ring-down technique, which will be described in more detail below.

According to the present invention, it is particularly advantageous to provide an additional reference chamber 22 in one and the same chip. Preferably, the training takes place mirror-symmetrically along 1 Dotted lines labeled YY and XX. According to the in 1 indicated arrows in and out of the supply and discharge channels 21a . 21b ; 22a . 22b becomes the actual measuring chamber 21 from a carrier liquid to which the analyte to be determined is flowed through. Should be in the reference chamber 22 identical immobilized nanoparticle layers are provided (which is not the case in the example shown), the otherwise identically designed reference measuring chamber 22 only flows through the otherwise unspecified carrier liquid here. Only by a lateral displacement of the Durflussküvette 1 What can be done by conventional optical adjustment within the CRD measuring device described below easily in the same operation and especially without any influence on measurement paths or even separate reference measurements (as in the prior art in conventional cuvettes usual) is thus the reference measuring chamber 22 in the beam path of the Messapperatur spending bar (see. 2 dotted thick arrow).

It is also within the scope of the invention, only one chamber 21 to provide, which also serves as a measuring and reference chamber with appropriate design and training. The only proviso, while complying with all other conditions, is that a larger expansion of the measuring chamber is selected in one direction (in the example y) than the smallest chamber expansion to be observed (in the example x) and in this direction (y) one of nanoparticles ( 12 ) Occupancy-free reference surface is provided. If a reference measurement is then carried out to determine the losses caused solely by the nanoparticles, the chamber is initially only flowed through by the carrier liquid and the reference measurement is carried out by shifting the chamber in the y direction in the area free of nanoparticles. Likewise, in the case of such a chamber formation with considerably larger chamber extent in one direction than prescribed by the other provisos of the present invention, it is within the scope of the invention that several differently prepared coatings with nanoparticles (US Pat. 12 ) are provided in one or more measuring chambers, with which, for example, different analytes or analyte mixtures, which dock on differently immobilized nanoparticles, can be examined in one operation.

According to the object of the present invention is thus a microfluidic flow cell 1 created that allows a highly sensitive detection of the changes in the absorption, respectively trans mission properties of a sample in the microfluidic system with very low optical density even at the very short optical path lengths, due to the very small sample volumes in the microfluidic system. Minimal changes in the transmission properties of the microfluidic system due to the sensor-active nanoparticles 12 , are detectable here.

In particular, changes in the absorption spectrum (amplitudes and spectral position, cf. 4 ) after binding of analyte molecules to the intended nanoparticles 12 be reliably detected in order to derive quantitative measures for corresponding sensors, eg. For the determination of DNA sequences derived. At low concentrations of the analyte molecules only very small shifts or absorption changes are to be expected, which in principle can not be detected with absorption spectrometers according to the prior art (see above).

In the proposed solution, the measurement signal is ideally proportional z. As to the concentration or the coverage of the analyte. The created according to the invention microfluidic flow cell 1 besides the CRD arrangement in which it is placed, it has a much smaller size than prior art spectrometers, and has the potential for further miniaturization.

• • die Messung von deutlich geringeren Absorptionsänderungen (2–3 Größenordnungen), als mit Zweistrahlspektrometern (>1 Promille, s. o.) nach dem Stand der Technik, wird hiermit möglich;
• • die dabei erzielbare hohe Genauigkeit und kompakte Bauform ermöglicht Messungen Vor-Ort, d. h. außerhalb von Laborbedingungen;
• • online/real-time-Messung auch während des Experiments sind möglich, da die Messkammer bei der Messung (sprich: bei der Analytzuführung) keine sonstigen Veränderungen erfährt.

The main advantages of the present invention are, inter alia, in the following:

• • The measurement of significantly lower absorption changes (2-3 orders of magnitude), as with two-beam spectrometers (> 1 per thousand, so) according to the prior art, is hereby possible;
• • The achievable high accuracy and compact design allows on-site measurements, ie outside of laboratory conditions;
• • Online / real-time measurement is also possible during the experiment, as the measuring chamber does not undergo any other changes during the measurement (ie during analyte delivery).

The sensor principle, ie the analyte molecules produce an absorption change on the plasmonically active nanoparticles 12 , Based on the highly sensitive measurement method - the cavity-ring-down method - for their application, the inventive microfluidic flow cell 1 is specially designed. Methodically, this involves a change from the measurement of the transmission (as in the case of "absorption spectrometers" according to the prior art) to the measurement of lowest losses which occur composed of all occurring absorptions, reflections and scatters. The measurand is no longer directly a change in intensity, but the average "lifetime" of photons in an empty laser resonator.

• • Die hier für die CRD Anwendung entwickelte Mikrofluid-Durchflussküvette besteht im Gegensatz zu üblichen Mikrofluidikchips aus zwei Chiphälften – bevorzugt einem Quarzglaswafer 2 mit definiert geätzten Strukturen im Durchstrahlbereich (vgl. 1) und einem Quarzglasdeckplättchen (vgl. 2), auf dem die plasmonisch aktiven Partikel 12 (z. B. Goldpartikel) aufgebracht sind. Die . Oberflächenbelegungsdichte der aufgebrachten Nanopartikel pro cm² ist dabei so gering zu halten, dass der im CRD Verfahren gemessene Verlust von Mikroküvette mit Nanopartikeln nur gering von dem der leeren Mikroküvette abweicht. Da dieser Verlust (u. a. Absorption) sehr stark von Material, Größe und Form der plasmonisch aktiven Nanopartikel abhängt, kann hier kein konkreter Zahlenwert genannt werden. Entscheidend ist, dass die Belegung mit Nanopartikeln 12 so vorgenommen wird, dass der durch sie verursachte Verlust in jedem Fall unter 1 Promille liegt, damit die Ring-down-Zeiten im (gut messbaren) μs-Zeitbereich liegen. Die theoretischen Grundlagen für die Dimensionierung einer Cavity und damit der Abklingzeiten werden in [ G. Berden, R. Engeln: Cavity ring down spectroscopy – Techniques and Applications, Wiley 2009 , ISBN: 978-1-4051-7688-0 ] (Resonatortheorie) beschrieben bzw. werden auch im Weiteren näher dargelegt.
• • Die Verbindung beider Chipkomponenten ist im Rahmen der Erfindung reversibel gestaltet und kann durch eine Fügesubstanz, z. B. durch Klebefolie, adhäsive chemische Substanzen, thermisch lösbare Substanzen oder dgl. hergestellt werden. Durch diesen Aufbau können alle Komponenten der Mikrofluid-Durchflussküvette nach Reinigung wiederverwendet und ggfs. an geänderte Messanforderungen angepasst werden.
• • Die durch mikrosystemtechnische Ätzprozesse hergestellte Struktur (1), besteht aus einer Messkammer 21, deren laterale Mindest-Ausdehnung in x-y-Richtung durch den Lichtstrahldurchmesser d_L (hier definiert durch die 1/e Breite der Intensitätsverteilung des Laserstrahls am Probenort innerhalb der CRD-Anordnung) festgelegt wird. Die Apertur der Mikrofluid-Durchflussküvette 1 am Messort soll erfindungsgemäß in den beiden Richtungen (x, y; vgl. 1) senkrecht zur Strahlungsrichtung L mindestens den dreifachen Durchmesser d_L des Laserstrahls (bei der 1/e Breite) aufweisen. Im speziellen Beispiel weisen die Krümmungsradien der Resonatorspiegel 3 (vgl. 3) z. B. r = 1000 mm und einen Spiegelabstand von 400 mm auf. Daraus folgt aus der Resonatortheorie ein minimal möglicher Strahldurchmesser (Grundmode der Cavity) für eine Wellenlänge von 650 nm von 0,575 mm. Diese Größe legt die vorstehend genannte Aperturbreite fest und muss demnach mindestens 1,725 mm betragen. Entsprechend ist die Mikrofluid-Durchflussküvette 1, d. h. im Besonderen die Berandungen der Kammern 21, 22 in diesem Beispiel ausgeführt. Die Dicke der Kammerwände und die Probendicke sind grundsätzlich zunächst frei wählbar. Generell sollten die Wände und die Probendicke jedoch so gering wie möglich (im speziellen Beispiel: 0,7 mm für die Dicke des Glaswafers 2; h = 60 μm) gewählt werden, damit sowohl das Probenvolumen als auch die resultierenden Absorptionen (Verluste) der Anordnung ohne Nanopartikel gering gehalten werden können.
• • Die Präparation des Plättchens 11, eine sehr dünne Glasplatte (typische Dicke: 200 μm), mit den Nanopartikeln 12, kann definiert vorgenommen werden, da das Plättchen erst nach der Präparation mit der anderen Chiphälfte über die Fügesubstanz 13 reversibel verbunden wird. Somit ist gewährleistet, dass die Mikrofluid-Durchflussküvette 1 mit ihren Funktionen, z. B. verschiedene Partikelkonzentrationen, optimal auf die experimentellen Bedürfnisse angepasst werden kann. Die Präparation kann jederzeit abgebrochen und neu begonnen werden, ohne die strukturierte Chiphälfte 2 zu belasten.
• • Die so speziell vorgefertigte Mikrofluid-Durchflussküvette 1 wird senkrecht zur Strahlausbreitung (vgl. 3) in den Resonatorraum der CRD-Anordnung eingebracht, um Zusatzverluste durch Reflexion in der Cavity zu unterbinden. Das senkrecht reflektierte Licht bleibt in der Cavity, bei anderen Winkeln würden Zusatzverluste auftreten. Diese Maßgabe setzt voraus, dass die Mikroküvettenwände planparallel gefertigt sind – typische Spezifikation bei der Optikfertigung von 5 Bogenminuten haben sich dabei als ausreichend erwiesen.
• • Die optischen Eigenschaften der Nanosensoren 12 werden durch ihr Material, Form und Größe exakt definiert und zeichnen sich durch ein charakteristisches Extinktionsspektrum aus (s. o.). Für eine optimale Detektion der spektralen Extinktionsänderung ist die Laserwellenlänge und der Cavity-Ring-Down Aufbau hierauf abzustimmen. Durch Wahl der Laserwellenlänge und/oder der spezifischen Nanopartikel kann man z. B. wählen, ob man im Maximum (hohes Signal) oder in der Flanke des Spektrums (hohe Empfindlichkeit gegen Absorptionsänderungen) detektiert. – Beispiele für Nanopartikel 12 sind: – Gold-Nanopartikel, sphärische Form, Durchmesser 30 nm: – Absorptionsmaximum bei 530 nm mit ca. 100 nm Halbwertsbreite – Silber(Ag)-Nanopartikel, sphärische Form, Durchmesser 50 nm: Absorptionsmaximum bei 530 nm mit ca. 80 nm Halbwertsbreite – Bei nicht-sphärischer Form (z. B. dreieckig) der Nanopartikel 12 verschieben sich die Spektren zum langwelligen Spektralbereich – Als Materialien für Nanopartikel mit den gewünschten Eigenschaften sind unter anderem bekannt und für die hier beschriebene Verwendung entsprechend geeignet: Au, Ag, Pt, Ca.
• Zur Verdeutlichung des Vorstehenden zeigt 4 eine beispielhafte Verschiebung des Absorptionsspektrums von Goldpartikeln (in diesem speziellen Beispiel) um Δλ von ca. 20 nm und eine in der Flanke dadurch bedingte Änderung der Extinktion ΔE von 0,0005 mit und ohne Bindung eines Analyten (im Beispiel einer DNA-Sequenz). In diesem speziellen Beispiel sollte der Nachweis von Phytophthora Kernoviae erfolgen. Vermittels lithographischer Strukturierung wurden dazu Nanosensoren mit einer Dichte von 0,1 Nanopartikeln/μm² aus Goldpartikeln mit Abmessungen 100·100·20 nm³, die ein Extinktionsmaximum in Wasser bei 690 nm aufweisen, gebildet. Ein Diodenlaser mit 650 nm Wellenlänge fand Verwendung. Die Resonatorspiegel 3, 31 in der jeweils verwendeten Cavity wurden im Bereich von 610–690 nm hochreflektierend (R > 99,98%) gewählt. Der Verlust der gesamten Mikrofluid-Durchflussküvette (vgl. rechte Kammerhälfte in 2, d. h. ohne Nanosensoren) betrug dabei 0,18%. Der Verlust, der allein durch die Anwesenheit der Nanosensoren 12 in Wasser entstand (vgl. linke Kammer 21 in 2) abzüglich des Verlusts durch die übrigen durchstrahlten Kammerwandungen, lag bei 0,197%. Der Verlust, der durch Andocken der zu untersuchenden DNA an die immobilisierten Nanopartikel 12 (Phytophthora Kernoviae Capture) zusätzlich hervorgerufen wurde, betrug 0,04%, während der Verlust durch den andockenden Analyten (hier: Phytophthora Kernoviae) bei 0,075% lag.
• • In 3 sind beispielhaft mögliche Messplatzvarianten dargestellt, in die die Mikrofluid-Durchflussküvette 1 einbringbar ist. Dabei stehen die einzelnen Beispiele A bis C für: A – Messplatz für eine monochromatische Anregung und Detektion B – Messplatz mit spektral breitbandiger Anregung und Detektion C – Weiteres Ausführungsbeispiel für eine andere Resonatorgeometrie mit geringerem Strahlquerschnitt am Ort des Mikrofluidikchip. Weitere Resonatoranordnungen liegen im Rahmen der Erfindung, wesentlich ist nur die Positionierung der Mikrofluid-Durchflussküvette 1 im Wesentlichen in oder in der Nähe der Strahltaille bei Einhaltung der Maßgabe, dass d_L mindestens ein Drittel der lateralen Mindestkammerausdehnung senkrecht dazu beträgt. Entsprechend der Ausführungen nach A und C kann ein gepulster Laser L als Strahlungsquelle oder nach den Ausführungen nach B und C eine gepulste Weißlichtquelle 6 (z. B eine entsprechende Lampe oder ein Faserlaser λ = 350 – 2600 nm) als Lichtquelle Verwendung finden. Den Resonatorraum bilden Laserspiegel mit einer hoch reflektiven Beschichtung (R > 99%). In der jeweils gebildeten Strahltaille im Resonatorraum, wird die entsprechend zu dimensionierende Mikrofluid-Durchflussküvette 1, entsprechend genannter Maßgaben so platziert, dass entweder die Messkammer 21 oder die Referenzkammer 22 (respektive die als Referenz dienende Fläche innerhalb der Messkammer) dort mittig vom Strahlungsfeld 5 erfasst wird. Je nach spezieller Ausführung erfolgt die Messwertaufnahme mittels eines Detektors 4 für eine monochromatische Detektion des CRD Signals oder mittels eines Detektorsystems 7 mit Spektrometer zur Wellenlängenselektierung und z. B. gatebarer (= zeitlich schnell schaltbarer) CCD zur zeitlichen Messwertaufnahme. Die Reflektivitäten der eingesetzten Resonatorspiegel 3, 31 sollen so gewählt sein, dass die durch sie verursachten optischen Verluste im Bereich der durch die Nanopartikel bedingten Absorption, d. h. da, wo die Nanopartikelsensoren ihr Extinktionsmaximum aufweisen, möglichst gering sind. Damit wird die spektrale Empfindlichkeit der Gesamtanordnung inklusive der Mikrofluid-Durchflussküvette 1 (vgl. 3) maximiert. Als Anregungslichtquellen kommen bevorzugt entweder monochromatische oder spektral breitbandige gepulste Laser in Frage. Da beim CRD-Verfahren generell nur Abklingkurven ausgewertet werden, ist auch der Einsatz von Dauerstrichlasern möglich, deren Emission mittels eines schnellen Schalters (z. B. elektro-optische oder akusto-optische Schalter) abgeschaltet bzw. abgelenkt werden kann. Die Detektionssysteme 4, 7 werden auf die jeweilige eingesetzte Lichtquelle bezüglich ihrer spektralen Empfindlichkeit angepasst. Für den Fall in Abbildung B wurde zusätzlich ein Spektrometer mit einer schnellen (im ns-Bereich) schaltbaren CCD Kamera für die Wellenlängenseparation verwendet. Fall C in 3 zeigt eine Anordnung, um mittels der größeren Freiheitsgrade eines Drei-Spiegel-Resonators (drei Spiegelkrümmungen, zwei Abstände) auch kleinere Laserstrahldurchmesser (d_L = 20 μm sind erreichbar) zu realisieren. Dies sollte nur beispielhaft erwähnt werden, um anzudeuten, dass durch eine solche oder weitere Miniaturisierungsmaßnahmen eine weitergehende Verkleinerung der Gesamtmessvorrichtung im Rahmen vorliegender Erfindung liegt, solange die Maßgabe eingehalten wird, dass der 1/e-Lichtstrahldurchmesser am Messort nicht größer als ein Drittel der geringsten lateralen Kammerwandbegrenzung beträgt. Das heißt, kleinere Resonatorgeometrien mit kleinerer Strahltaille d_L am Messort in der Mikrofluid-Durchflussküvette 1 ermöglichen auch kleinere mögliche Geometrien der Küvette selbst, respektive der Messkammer. Ebenso sind im Rahmen der Erfindung auch faseroptische Ausführungen denkbar, bei denen die Resonatorwirkung und Strahlformung durch entsprechend gestaltete Lichtleitfasern übernommen werden, ohne vorstehende Maßgaben an die erfindungsgemäße Gestaltung der Mikrofluid-Durchflussküvette zu verändern.
• • Betrachtungen zu den auftretenden Verlusten: Um die sensorische Aktivität der plasmonisch aktiven Nanopartikel 12 in einer CRD Anordnung gut nachweisen zu können, müssen diese in niedriger Konzentration (s. o.) in der Mikrofluid-Durchflussküvette 1 integriert werden. Die exakte Oberflächenbelegungsdichte der Nanosensoren ist, wie bereits erwähnt, von den verwendeten Nanopartikeln (Material, Form, Größe) abhängig. Der zusätzliche optische Verlust, den nur die Nanosensoren verursachen, sollte für eine hohe Genauigkeit im Bereich der optischen Verluste der CRD Anordnung mit Mikrofluidikchip (aber ohne Nanopartikel) liegen. Die Verluste des gesamten Aufbaus setzen sich zusammen aus:

  T

  Transmission aller Resonatorspiegel,

  S

  Streuung an und in allen durchstrahlten Medien (außer Nanopartikeln),

  A_ref

  Absorption in allen durchstrahlten Medien (außer Nanopartikel) und

  A

  optischer Verlust, bedingt durch Absorption und Streuung an den Nanopartikeln.

For this purpose, according to the invention, the microfluidic system (that is, the microfluidic flow-through cuvette through which the analyte can flow) and a standard structure for CRD measurement are matched to one another. The following points are to be considered in the special design of the entire measurement setup in the context of the invention in principle:

• • In contrast to common microfluidic chips, the microfluidic flow cell developed here for the CRD application consists of two half-tubes - preferably a quartz glass wafer 2 with defined etched structures in the transmission area (cf. 1 ) and a quartz glass cover plate (cf. 2 ) on which the plasmonically active particles 12 (eg gold particles) are applied. The . Surface coverage density of the applied nanoparticles per cm ² is to be kept so low that the loss of microcuvette with nanoparticles measured in the CRD method deviates only slightly from that of the empty microcuvette. Since this loss (including absorption) depends very much on the material, size and shape of the plasmonically active nanoparticles, no concrete numerical value can be mentioned here. What matters is that the occupancy of nanoparticles 12 is made in such a way that the loss caused by them in any case is less than 1 per thousand, so that the ring-down times are in the (well measurable) μs time range. The theoretical basis for the dimensioning of a cavity and thus the cooldowns are given in [ G. Berden, R. Engeln: Cavity ring down spectroscopy - Techniques and Applications, Wiley 2009 . ISBN: 978-1-4051-7688-0 ] (Resonator theory) are described or will be explained in more detail below.
• • The connection of both chip components is designed reversible in the context of the invention and can by a joining substance, for. B. by adhesive film, adhesive chemical substances, thermally soluble substances or the like. Thanks to this design, all components of the microfluidic flow cell can be reused after cleaning and, if necessary, adapted to changing measurement requirements.
• The structure produced by microsystem etching processes ( 1 ), consists of a measuring chamber 21 whose lateral minimum extent in the xy direction is determined by the light beam diameter d _L (defined here by the 1 / e width of the intensity distribution of the laser beam at the sample location within the CRD arrangement). The aperture of the microfluidic flow cell 1 At the measuring location according to the invention, in the two directions (x, y; 1 ) perpendicular to the radiation direction L have at least three times the diameter d _{L of} the laser beam (at the 1 / e width). In the specific example, the radii of curvature of the resonator mirror 3 (see. 3 ) z. B. r = 1000 mm and a mirror spacing of 400 mm. It follows from the resonator theory a minimum possible beam diameter (fundamental mode of the cavity) for a wavelength of 650 nm of 0.575 mm. This size defines the above-mentioned aperture width and must therefore be at least 1.725 mm. Accordingly, the microfluidic flow cell 1 , ie in particular the boundaries of the chambers 21 . 22 in this example. The thickness of the chamber walls and the sample thickness are basically initially freely selectable. Generally, however, the walls and the sample thickness should be as low as possible (in the specific example: 0.7 mm for the thickness of the glass wafer 2 ; h = 60 μm), so that both the sample volume and the resulting absorptions (losses) of the arrangement without nanoparticles can be kept low.
• • The preparation of the platelet 11 , a very thin glass plate (typical thickness: 200 μm), with the nanoparticles 12 , can be made defined, since the plate only after the preparation with the other half of the chip on the joining substance 13 is reversibly connected. This ensures that the microfluidic flow cell 1 with their functions, eg. B. different particle concentrations can be optimally adapted to the experimental needs. The preparation can be stopped at any time and restarted without the structured half of the chiphase 2 to charge.
• • The specially pre-fabricated microfluidic flow cell 1 becomes perpendicular to the beam propagation (cf. 3 ) are introduced into the resonator chamber of the CRD arrangement in order to prevent additional losses due to reflection in the cavity. The vertically reflected light remains in the cavity, at other angles additional losses would occur. This requirement presupposes that the microcuvette walls are manufactured plane-parallel - typical specification in the optical production of 5 arc minutes have proven to be sufficient.
• • The optical properties of nanosensors 12 are defined exactly by their material, shape and size and are characterized by a characteristic extinction spectrum (see above). For optimum detection of the spectral change in extinction, the laser wavelength and the cavity-ring-down structure must be adapted to this. By choosing the laser wavelength and / or the specific nanoparticles can be z. For example, choose whether to be in the maximum (high signal) or in the Edge of the spectrum (high sensitivity to absorption changes) detected. - Examples of nanoparticles 12 are: - gold nanoparticles, spherical shape, diameter 30 nm: - absorption maximum at 530 nm with about 100 nm half-width - silver (Ag) nanoparticles, spherical shape, diameter 50 nm: absorption maximum at 530 nm with about 80 nm half-width - For non-spherical shape (eg triangular) of nanoparticles 12 the spectra shift to the long-wave spectral range. As materials for nanoparticles with the desired properties, among others, they are known and suitable for the use described here: Au, Ag, Pt, Ca.
• To clarify the above shows 4 an exemplary shift of the absorption spectrum of gold particles (in this particular example) by Δλ of about 20 nm and an edge-related change in the extinction ΔE of 0.0005 with and without binding of an analyte (in the example of a DNA sequence). In this particular example, evidence of Phytophthora kernoviae should be provided. By means of lithographic structuring, nanosensors having a density of 0.1 nanoparticles / μm ² of gold particles with dimensions of 100 × 100 × 20 nm ³ and having an extinction maximum in water at 690 nm were formed for this purpose. A diode laser with 650 nm wavelength was used. The resonator mirrors 3 . 31 in the cavity used in each case highly reflective (R> 99.98%) were selected in the range of 610-690 nm. The loss of the entire microfluidic flow cell (see right half of the chamber in 2 , ie without nanosensors) was 0.18%. The loss caused solely by the presence of nanosensors 12 was formed in water (see left chamber 21 in 2 ) minus the loss through the other irradiated chamber walls, was 0.197%. The loss caused by docking of the DNA under investigation to the immobilized nanoparticles 12 (Phytophthora Kernoviae Capture) was 0.04%, while the loss from the docking analyte (here: Phytophthora Kernoviae) was 0.075%.
• • In 3 exemplary possible measuring station variants are shown, in which the microfluidic flow cell 1 can be introduced. Here, the individual examples A to C stand for: A - measuring station for a monochromatic excitation and detection B - measuring station with spectrally broadband excitation and detection C - further exemplary embodiment of another resonator geometry with a smaller beam cross section at the location of the microfluidic chip. Further resonator arrangements are within the scope of the invention; only the positioning of the microfluid flow cell is essential 1 substantially in or near the beam waist, while maintaining the proviso that d _{L is} at least one third of the minimum lateral chamber extent perpendicular thereto. According to the embodiments according to A and C, a pulsed laser L as a radiation source or according to the embodiments according to B and C, a pulsed white light source 6 (For example, a corresponding lamp or a fiber laser λ = 350 - 2600 nm) find use as a light source. The resonator cavity is formed by laser mirrors with a highly reflective coating (R> 99%). In the respectively formed beam waist in the resonator chamber, the correspondingly dimensioned microfluidic flow cuvette becomes 1 , according to specified specifications placed so that either the measuring chamber 21 or the reference chamber 22 (respectively the area serving as a reference within the measuring chamber) there in the middle of the radiation field 5 is detected. Depending on the specific version, the measured value is recorded by means of a detector 4 for a monochromatic detection of the CRD signal or by means of a detector system 7 with spectrometer for wavelength selection and z. B. gateable (= temporally fast switchable) CCD for temporal data acquisition. The reflectivities of the resonator mirrors used 3 . 31 should be chosen so that the optical losses caused by them in the range of the absorption caused by the nanoparticles, ie, where the nanoparticle sensors have their Absinmaximum, are as low as possible. This adjusts the spectral sensitivity of the overall assembly, including the microfluidic flow cell 1 (see. 3 ) maximizes. As excitation light sources are preferably either monochromatic or spectrally broadband pulsed laser in question. Since the CRD method generally evaluates only decay curves, it is also possible to use continuous wave lasers whose emission can be switched off or deflected by means of a fast switch (eg electro-optical or acousto-optical switches). The detection systems 4 . 7 are adapted to the particular light source used with regard to their spectral sensitivity. In the case of Figure B, a spectrometer with a fast (in the ns range) switchable CCD camera was used for the wavelength separation. Case C in 3 shows an arrangement to realize by means of the greater degrees of freedom of a three-mirror resonator (three mirror curvatures, two distances) and smaller laser beam diameter (d _L = 20 microns are achievable). This should be mentioned only by way of example in order to indicate that, by means of such or further miniaturization measures, a further reduction of the total measuring device in the context of the present invention as long as it is complied with that the 1 / e-beam diameter at the measuring location does not exceed one third of the lowest lateral chamber wall limit. That is, smaller resonator geometries with smaller beam waist d _L at the measurement location in the microfluidic flow cell 1 also allow smaller possible geometries of the cuvette itself, respectively the measuring chamber. Likewise, within the scope of the invention, fiber optic designs are also conceivable in which the resonator effect and beam shaping are taken over by appropriately designed optical fibers without modifying the above specifications to the design of the microfluid flow cell according to the invention.
• • Considerations to the occurring losses: To the sensory activity of the plasmonic active nanoparticles 12 In order to be able to detect well in a CRD arrangement, these must be in low concentration (see above) in the microfluidic flow cell 1 to get integrated. As already mentioned, the exact surface occupation density of the nanosensors depends on the nanoparticles used (material, shape, size). The additional optical loss that only the nanosensors cause should be for high accuracy in the optical loss range of the microfluidic chip (but without nanoparticle) CRD assembly. The losses of the entire structure are composed of:

  T

  Transmission of all resonator mirrors,

  S

  Scattering on and in all irradiated media (except nanoparticles),

  A _ref

  Absorption in all irradiated media (except nanoparticles) and

  A

  optical loss due to absorption and scattering at the nanoparticles.

The surface occupation density of the nanoparticles 12 is chosen according to these provisos so that for the optical loss A, which is produced only by the nanoparticles, the following applies: 0.1 * [T + S + A _ref ] <A <10 * [T + S + A _ref ]

The above-mentioned range is that in which still sufficiently large measurement signals are obtained. However, the ratios are preferably to be selected so that the losses caused by the arrangement with a microfluidic chip (but without nanoparticles) are of the same order of magnitude as that caused by the nanoparticles 12 caused. With this nanoparticle concentration, the function of the measuring method is ensured and at the same time the maximum possible sensitivity of the test arrangement is achieved.

It is also within the scope of the invention to arrange a plurality of different microfluidic flow cells side by side in series or parallel with respect to the flow separated from each other in a wafer or, as already mentioned above, to provide a plurality of separately prepared nanoparticle sensors in a measuring chamber of the microfluidic flow cell.

LIST OF REFERENCE NUMBERS

1

Microfluidic flow cell

11

optically transparent plate

12

nanoparticles

13

Detachable connection

2

wafer

21

Flow measurement chamber

21a / 21b

Inflow and outflow channel to the measuring chamber

22

reference chamber

22a / 22b

Inflow and outflow channel to the reference chamber

3, 31

resonator

4

Detector for monochromatic detection

5

radiation field

6

White light source

7

Detector system with spectrometer

d _l

Beam diameter at maximum intensity · 1 / e

L

beam of light

X-X, Y-Y

cutting planes

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102006035581 B3 [0005]

Cited non-patent literature

• Mark, et al .: Chem. Soc. Rev., 39, 1153-1182, (2010) [0002]
• VG Kutchoukov, et al .: Sensors and Actuators A, 114, 1521-527, (2004) [0002]
• Tapan K. Sau, et al .: Advanced Materials 22, 16, 1805-1825, (2010) [0003]
• G. Berden, R. Peeters, G. Meijer; Cavity ring-down spectroscopy; Int. Rev. Phys. Chem. 19, 565-607 (2000) [0007]
• G. Schmidl, W. Paa, W. Triebel, St. Schippel, H. Heyer; Appl. Optics, Vol. 48, No 35; (2009) [0007]
• G. Berden, R. Engeln: Cavity ring down spectroscopy - Techniques and Applications, Wiley 2009 [0007]
• ISBN: 978-1-4051-7688-0 [0007]
• Hans-Peter Loock: Trends in Analytical Chemistry, Vol. 7, 2006 [0007]
• Van der Sneppen, F. Ariese, C. Gooijer, W. Ubachs: Annu. Rev. Anal. Chem. 2009. 2: 13-35 [0007]
• G. Berden, R. Engeln: Cavity ring down spectroscopy - Techniques and Applications, Wiley 2009 [0007]
• ISBN: 978-1-4051-7688-0 [0007]
• Hans-Peter Bock: Trends in Analytical Chemistry, Vol. 7, 2006 [0007]
• Van der Sneppen, F. Ariese, C. Gooijer, W. Ubachs: Annu. Rev. Anal. Chem. 2009. 2: 13-35 [0007]
• G. Berden, R. Engeln: Cavity ring down spectroscopy - Techniques and Applications, Wiley 2009 [0025]
• ISBN: 978-1-4051-7688-0 [0025]

Claims (10)

Microfluidic flow cell ( 1 ) for use in a cavity ring-down (CRD) absorption spectroscopy system for detecting lowest analyte concentrations, in which an optically transparent wafer ( 2 ) at least one flow measuring chamber ( 21 ) with inflow and outflow channel ( 21a . 21b ), wherein the actual measuring chamber in the region of the vertical transmission with a light beam (L) in the beam waist of the CRD arrangement, in which the intensity of the light beam has fallen to 1 / e (d _L ), an extension (x, y ) such that the smallest chamber extent (x, y) in this area is at least three times the light beam diameter (d _L ) and the wafer ( 2 ) with one the chamber ( 21 ) overlapping optically transparent platelets ( 11 ) is connected in a planar manner, whereby the chamber ( 21 ) facing surface at least in the region of the intended light transmission with at least one surface with immobilized nanoparticles ( 12 ) and by wafers ( 2 ) and platelets ( 11 ) conditional total height (h _g ) is set parallel to the light transmission direction of the flow cell below one millimeter. Microfluidic flow cell according to claim 1, characterized in that in the wafer ( 2 ) a second identical to the measuring chamber ( 21 ) executed reference chamber ( 22 ) is provided. Microfluidic flow cell according to claim 2, characterized in that the reference chamber ( 22 ) with its inflow and outflow channel ( 22a . 22b ) mirror-symmetrical to the measuring chamber ( 21 ) is arranged. Microfluidic flow cell according to claim 1, characterized in that the wafer ( 2 ) outside the borders of the chambers ( 21 . 22 ) and respective inflow and outflow channels ( 21a . 21b ; 22a . 22b ) with the plate ( 11 ) is connected tightly but detachably. Microfluidic flow cell according to claim 4, characterized in that the tight and detachable compound ( 13 ) is formed by a joining agent such as an adhesive sheet, a thermally releasable adhesive or an otherwise adhesive releasable bonding agent. Microfluidic flow cell according to one of the preceding claims, characterized in that the through the wafer ( 2 ), the connecting means ( 13 ) and the glass plate ( 11 ) formed layer system is carried out plane-parallel. Microfluid flow cell according to claim 1, characterized in that the surface occupation density of the provided assignments with immobilized nanoparticles ( 12 ) is defined and adjustably set by the nanoparticles ( 12 ) conditional optical loss A when the flow cell is moved into a cavity ring-down arrangement in a range 0.1 · [T + S + A _ref ] <A <10 · [T + S + A _ref ], where T for the transmission of all resonator mirrors. S for the scattering on and in all irradiated cuvette components including the fluid flowing through, except at the nanoparticles and A _ref for absorption in all irradiated cuvette components including the fluid flowing through, except at the nanoparticles. Microfluidic flow cell according to claim 7, characterized in that the surface occupation density of the provided assignments with immobilized nanoparticles ( 12 ), its shape, size and choice of material are defined so that the losses caused by the analyte are of the same order of magnitude as that which is determined by the nanoparticles ( 12 ) caused itself. Microfluidic flow cell according to claim 1, characterized in that the height (h) of the chambers ( 21 . 22 ) is set as low as possible depending on the viscosity of the carrier liquid, but so large that a flow through the chamber is ensured with liquid, which is advantageously given in a range of a few 10 microns to a few 100 microns, this height (h) but always very small compared to the lateral chamber expansions (x, y) is selected. Microfluidic flow cell according to claim 1 and 9, characterized in that with a considerably larger expansion of the measuring chamber in one direction (y) than the smallest chamber extent (x) to be maintained, in this direction (y) several differently prepared coatings with nanoparticles ( 12 ) and / or at least one of nanoparticles ( 12 ) occupancy-free reference surface are provided.

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