EP1672674A1 - Méthode et système pour l'analyse de masses à haut-débit - Google Patents

Méthode et système pour l'analyse de masses à haut-débit Download PDF

Info

Publication number
EP1672674A1
EP1672674A1 EP04030063A EP04030063A EP1672674A1 EP 1672674 A1 EP1672674 A1 EP 1672674A1 EP 04030063 A EP04030063 A EP 04030063A EP 04030063 A EP04030063 A EP 04030063A EP 1672674 A1 EP1672674 A1 EP 1672674A1
Authority
EP
European Patent Office
Prior art keywords
sample
jet
microfluidic
samples
examination
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04030063A
Other languages
German (de)
English (en)
Inventor
Jürgen TROE
Ales Charvat
Bernd Abel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Georg August Universitaet Goettingen
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Georg August Universitaet Goettingen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften eV, Georg August Universitaet Goettingen filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority to EP04030063A priority Critical patent/EP1672674A1/fr
Priority to US11/721,832 priority patent/US8009287B2/en
Priority to PCT/EP2005/056835 priority patent/WO2006064048A1/fr
Publication of EP1672674A1 publication Critical patent/EP1672674A1/fr
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components

Definitions

  • the invention relates to an investigation method and a corresponding investigation system, in particular for the mass spectroscopy of biomolecules, according to the preamble of the independent claims.
  • the samples to be examined are dissolved in water as a carrier liquid, wherein the carrier liquid is injected with the sample dissolved therein through a micro nozzle in a vacuum, so that in the vacuum forms a stable micro liquid jet, which has a beam diameter in the range of 5-100 microns and a jet velocity of 30-100 m / s.
  • micro liquid jet molecules are then desorbed by irradiation with pulsed, tunable infrared laser light and then examined by mass spectroscopy.
  • This laser-induced desorption of sample molecules from the microfluidic jet advantageously enables a very gentle release of the sample molecules.
  • the known examination method described at the outset only permits the examination of a single sample substance which is dissolved in the carrier liquid of the microfluidic jet. To examine another sample substance, the carrier liquid must first be replaced with the old sample substance, which is extremely time-consuming.
  • the known examination method described at the outset therefore does not allow a high-throughput mass analysis of a large number of samples.
  • the invention is therefore based on the object to improve the initially described known examination method or the associated examination system accordingly.
  • the invention comprises the general technical teaching of introducing spatially limited samples into the carrier liquid of the microfluidic jet, each of which extends in the beam direction only over a partial region of the microfluidic jet.
  • a localized injection of the samples to be examined into the carrier liquid of the microfluidic jet advantageously makes it possible to rapidly change the samples to be analyzed by successively injecting the various samples into the microfluidic jet and examining them in succession.
  • the spatially limited injection of the samples significantly reduces the consumption of sample substance.
  • microfluidic stream In the case of a high-throughput mass analysis, several samples are preferably introduced into the microfluidic stream so that the individual samples in the microfluidic stream are arranged one behind the other in the jet direction and are spatially separated from one another.
  • the individual samples thus form plugs or segments in the microfluidic jet, which otherwise consists of the carrier liquid (for example water).
  • the injection of the individual samples into the carrier liquid can take place, for example, by means of a controllable valve, which is preferably arranged upstream of the micro nozzle which is used to produce a microfluidic jet.
  • the valve used may be, for example, a high pressure valve (HPLC valve), such as the Gynkotek Model 300C.
  • valves open in this case preferably in a carrier flow channel, which feeds the micro nozzle for generating the micro liquid jet.
  • the desorption of the individual samples from the microfluidic stream can be effected in a conventional manner by laser irradiation, for example by irradiation with pulsed, tunable, IR laser light, which is described in the document SPANGENBERG, Tim; A-BEL, Bernd: "Laser-excited microfilaments for extreme light sources and biomolecule analysis” as well as from CHARVAT, A. et al .: "New design for a time-of-flight mass spectrometer with a liquid beam desorption source for the analysis of biomolecules ", Review of scientific instruments, Volume 75, Number 5, May 2004, pages 1209 et seq.
  • the examination system according to the invention therefore preferably has a desorption device with a laser.
  • the examination of the samples desorbed from the microfluidic stream in the context of the invention can also be carried out in a conventional manner, for example by a mass-spectroscopic examination.
  • a mass-spectroscopic examination With regard to the examination of the samples desorbed from the microfluidic jet, reference is likewise made to the two aforementioned publications "Laser-excited microfilaments for extreme light sources and biomolecule analysis” and “New design for a time-of-flight mass spectrometer with a liquid beam.” the analysis of biomolecules ", the content of which is fully attributable to the present description in this context.
  • micro nozzle itself may be formed in the conventional manner in the context of the invention, such micro nozzles being described for example in the patent publication WO 2004/076071 A1.
  • the content of this patent publication is, therefore, to be fully attributed to the present specification.
  • the individual samples in the microfluidic jet may have a spatial extent in the beam direction which is so great that each sample can be hit several times by a laser pulse for desorption.
  • a multiple desorption of sample molecules from the individual samples allows, for example, a statistical evaluation of the resulting test results, for example by averaging.
  • the prerequisite for this, however, is that the product of the steel velocity and the desorption period (ie, as a rule, the period of the pulsed laser) must be smaller than the sample length of the individual samples so that a sample traveling with the microfluidic stream is successively detected by several laser pulses can.
  • the individual samples in the microfluidic jet have such a small sample length in the beam direction that each sample can only be detected by a single laser pulse.
  • the product of the jet velocity and the desorption period ie, the period duration of the pulsed laser light
  • Such short samples advantageously enable a mass throughput of a large number of samples with simultaneously low sample substance input (sample quantities).
  • the individual laser pulses must hit the individual samples exactly in order to effect a desorption of sample substance from the microfluidic jet. Therefore, within the scope of the invention, there is the possibility that the desorption (for example the laser pulses) is synchronized taking into account the sample length and the jet velocity, so that the individual laser pulses each hit exactly one of the samples.
  • Such a synchronization can for example be done passively by detecting the jet velocity and triggering the laser pulses as a function of the jet velocity.
  • the synchronization is active.
  • an optical barrier can be used for this purpose, through which the microfluidic jet passes, so that the individual samples can be detected as they pass through the optical barrier.
  • the delivery of the individual laser pulses can then be triggered in such a way that they exactly hit the individual samples.
  • the injection of the individual samples into the carrier liquid of the microfluidic jet preferably takes place upstream of the micronozzle, since there the flow rate of the carrier liquid is substantially lower than in the microfluidic stream downstream behind the micronozzle.
  • a sample magazine with a plurality of sample chambers can be used for the injection of the individual samples into the carrier liquid, wherein the individual sample chambers of the sample magazine can be loaded with the individual samples.
  • the sample magazine may then be inserted into the carrier flow line such that the carrier flow line flows through one of the sample chambers, carrying with it the sample substance contained therein.
  • the sample magazine may in this case be designed, for example, revolver-shaped and rotated accordingly during operation in order to successively introduce different samples into the carrier liquid.
  • the carrier liquid for receiving the samples to be examined may be, for example, conventional water.
  • the invention is not limited to water with respect to the carrier liquid to be used, but also with any other liquids feasible.
  • the individual samples have, for example, a sample volume in the range from 10 ⁇ l to 100 ml, with any intermediate values within this range being possible. Preferably, however, the sample volume is in the range of 10 ⁇ l to 100 ⁇ l.
  • the microfluidic jet preferably has a beam diameter in the range of 5 ⁇ m to 100 ⁇ m, whereby a range of 5 ⁇ m to 30 ⁇ m has proven to be particularly advantageous.
  • the microfluidic jet also has a jet velocity which is preferably in the range of 20 m / s to 200 m / s, with respect to the jet velocity Any intermediate values within the aforementioned range of values are also possible.
  • microfluidic jet between the micro-nozzle and its disintegration point where the microfluidic jet disintegrates into drops may contain a plurality of samples, such as more than 10, more than 50, or more than 100 samples.
  • the fast-flowing microfluidic jet between the micro-nozzle and the disintegration point i. in the so-called continuous area, containing only a single sample.
  • a plurality of samples can be examined in rapid succession, for example, more than 5 samples per second.
  • microfluidic jet is typically injected into a vacuum or vacuum chamber, with the desorption of the samples and / or the examination of the samples occurring in the vacuum chamber.
  • the invention also encompasses a microfluidic jet as such which contains spatially limited samples which extend in the beam direction only over a partial region of the microfluidic jet.
  • the schematic representation in Figure 1 shows a micro liquid jet 1, which has a beam diameter d in the range of 5 .mu.m to 100 .mu.m and a jet velocity vim range from 20 m / s to 200 m / s, wherein the micro liquid jet 1 by a known micro nozzle is injected into a vacuum and remains stable in the vacuum to a disintegration point, not shown, at which the microfluidic jet 1 then decomposes into droplets.
  • micro nozzle itself is in this case designed in a conventional manner according to the patent publication WO 2004/076071 A1, so that at this point a detailed description of the micro nozzle can be dispensed with.
  • a plurality of samples 2-4 in the form of a plug, wherein the samples 2-4 may contain different sample substances in order to enable a mass flow rate analysis of a plurality of samples.
  • the individual samples 2-4 are irradiated for desorption from the microfluidic beam 1 by an infrared laser 5 with laser pulses, which in itself from the above-cited references “laser-excited microfilaments for extreme light sources and biomolecule analysis” and “New design for a time It is known that reference is made to the publications in order to avoid repetition.
  • the infrared laser 5 outputs the individual laser pulses in this case with a period ⁇ t, which is adapted to the sample length L of the individual samples 2-4 such that the product of the jet velocity v and the pulse period is greater than the sample length L. This means that each of Samples 2-4 is hit only by a single laser pulse.
  • a special feature of this exemplary embodiment is that the sample length L of the individual samples 2, 3 is substantially greater than in the exemplary embodiment according to FIG. 1.
  • the product of the jet velocity v and the pulse period ⁇ t is smaller than the sample length L of the two samples 2, 3, so that each of the two samples 2, 3 is hit by several laser pulses.
  • several sample fragments are desorbed from each of the samples 2, 3 and examined separately. This allows a mean value of the examination results of the individual sample fragments.
  • the exemplary embodiment illustrated in FIG. 3 again largely corresponds to the exemplary embodiment described above and illustrated in FIG. 2, so that reference is again made to the above description of FIG. 2 in order to avoid repetition.
  • a special feature of this embodiment is that the delivery of the laser pulses is triggered by the infrared laser 5 by a synchronization device, so that the individual laser pulses meet exactly the samples 2, 3 respectively.
  • the embodiment has a light barrier, which consists of a laser 6 and an optical detector 7, wherein the laser beam emitted by the laser 6 passes through the micro liquid jet 1 and therefore allows detection of the individual samples 2, 3 when passing through the laser beam ,
  • the detector 7 controls the passage of the individual samples 2, 3 each have a control unit 8, which then the infrared laser 5 triggers so that the pulses emitted by this exactly hit the samples 2, 3.
  • FIGS. 4A and 4B show an injection device that can be used to inject the samples 2, 3 into the trigger liquid of the microfluidic jet 1.
  • the injection device is in this case arranged upstream of the micro nozzle, which injects the microfluidic jet 1 into the vacuum.
  • This arrangement is advantageous because the flow rate of the carrier liquid upstream of the micro-nozzle is substantially lower than that in micro-liquid jet 1 downstream of the micro-nozzle, thereby facilitating the injection of the samples 2, 3.
  • the injection device is arranged in the carrier flow line which feeds the micro nozzle, two carrier current line sections 9, 10 being illustrated in the drawing.
  • the carrier liquid is supplied in the injection device via the carrier flow line section 9 and leaves the injection device again via the carrier flow line section 10 to the micro nozzle, which injects the microfluidic jet 1 into the vacuum.
  • the carrier liquid flows through one of two sample chambers 11, 12 of a sample magazine 13, wherein the sample magazine 13 is rotatable in the arrow direction.
  • the carrier liquid flows via the carrier flow line section 9 through the sample chamber 11 into the carrier flow line section 10 and then on to the micro nozzle.
  • sample substance is introduced via a sample supply line 14 into the sample chamber 12 and flows through it in the direction of the sample outlet 15.
  • the sample magazine 13 can be rotated in the arrow direction, so that the sample chamber 12 filled with sample substance between the two carrier flow line sections 9, 10 and is therefore flushed with carrier liquid, wherein the sample chamber located in the sample chamber 12 Sample substance is carried.
  • the other sample chamber 11 can be filled with a new sample substance, which is shown in FIG. 4B.
  • FIGS. 5A and 5B show an alternative embodiment of an injection device for injecting the individual samples into the carrier liquid of the microfluidic jet 1.
  • This embodiment is partly identical to the embodiment described above and illustrated in FIGS. 4A and 4B, so that reference is made to FIGS. 4A and 4B in order to avoid repetition, the same reference numerals being used for corresponding components.
  • sample magazine 13 is revolver-shaped and can be rotated about an axis of rotation which is substantially parallel to the carrier power line sections 9, 10.
  • the individual sample chambers 16 thus form a coaxial component of the carrier power line sections 9, 10 in each case in one rotational position.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Engineering & Computer Science (AREA)
  • Plasma & Fusion (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
EP04030063A 2004-12-17 2004-12-17 Méthode et système pour l'analyse de masses à haut-débit Withdrawn EP1672674A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP04030063A EP1672674A1 (fr) 2004-12-17 2004-12-17 Méthode et système pour l'analyse de masses à haut-débit
US11/721,832 US8009287B2 (en) 2004-12-17 2005-12-15 Method and system for high throughput mass analysis
PCT/EP2005/056835 WO2006064048A1 (fr) 2004-12-17 2005-12-15 Procede et systeme d'etude destines a l'analyse de masse a debit eleve

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP04030063A EP1672674A1 (fr) 2004-12-17 2004-12-17 Méthode et système pour l'analyse de masses à haut-débit

Publications (1)

Publication Number Publication Date
EP1672674A1 true EP1672674A1 (fr) 2006-06-21

Family

ID=34927841

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04030063A Withdrawn EP1672674A1 (fr) 2004-12-17 2004-12-17 Méthode et système pour l'analyse de masses à haut-débit

Country Status (3)

Country Link
US (1) US8009287B2 (fr)
EP (1) EP1672674A1 (fr)
WO (1) WO2006064048A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110129717A (zh) * 2019-05-31 2019-08-16 上海大学 基于多源等离子喷涂和激光后处理的厚膜组合材料芯片高通量制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2017875A1 (fr) 2007-07-16 2009-01-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé et appareil pour fournir un échantillon pour une analyse successive

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4403147A (en) * 1979-05-25 1983-09-06 Hewlett-Packard Company Apparatus for analyzing liquid samples with a mass spectrometer
US6147344A (en) * 1998-10-15 2000-11-14 Neogenesis, Inc Method for identifying compounds in a chemical mixture
US20020072126A1 (en) * 2000-10-03 2002-06-13 Dionex Corporation Method and system for peak parking in liquid chromatography-mass spectrometer (LC-MS) analysis
US20040222373A1 (en) * 2003-04-29 2004-11-11 Sri International Direct liquid injection inlet to a laser photoionization apparatus

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1815958B1 (de) * 1968-12-20 1970-06-18 Zeiss Carl Fa Vorrichtung zur modulierten Zufuhr einer Probenfluessigkeit zu einer spektroskopischen Lichtquelle,z.B.einer Flamme
US3679312A (en) * 1971-06-16 1972-07-25 Technicon Instr Method and apparatus for measuring bioluminescence or chemiluminescence for quantitative analysis of samples
US6140639A (en) * 1998-05-29 2000-10-31 Vanderbilt University System and method for on-line coupling of liquid capillary separations with matrix-assisted laser desorption/ionization mass spectrometry
US6744041B2 (en) * 2000-06-09 2004-06-01 Edward W Sheehan Apparatus and method for focusing ions and charged particles at atmospheric pressure
DE10308299A1 (de) 2003-02-26 2004-09-16 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Düsenanordnung
US7247487B2 (en) * 2003-06-18 2007-07-24 Ortho-Clinical Diagnostics Reducing working fluid dilution in liquid systems

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4403147A (en) * 1979-05-25 1983-09-06 Hewlett-Packard Company Apparatus for analyzing liquid samples with a mass spectrometer
US6147344A (en) * 1998-10-15 2000-11-14 Neogenesis, Inc Method for identifying compounds in a chemical mixture
US20020072126A1 (en) * 2000-10-03 2002-06-13 Dionex Corporation Method and system for peak parking in liquid chromatography-mass spectrometer (LC-MS) analysis
US20040222373A1 (en) * 2003-04-29 2004-11-11 Sri International Direct liquid injection inlet to a laser photoionization apparatus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHARVAT, A., LUGOVOJ, E., FAUBEL, M., ABEL, B.: "New design for a time-of-flight mass spectrometer with a liquid beam laser desorption ion source for the analysis of biomolecules", REVIEW OF SCIENTIFIC INSTRUMENTS, vol. 75, no. 5, May 2004 (2004-05-01), pages 1209 - 1218, XP002321678 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110129717A (zh) * 2019-05-31 2019-08-16 上海大学 基于多源等离子喷涂和激光后处理的厚膜组合材料芯片高通量制备方法
CN110129717B (zh) * 2019-05-31 2021-11-02 上海大学 基于多源等离子喷涂和激光后处理的厚膜组合材料芯片高通量制备方法

Also Published As

Publication number Publication date
US8009287B2 (en) 2011-08-30
US20090321627A1 (en) 2009-12-31
WO2006064048A1 (fr) 2006-06-22

Similar Documents

Publication Publication Date Title
DE19822672B4 (de) Verfahren und Vorrichtung zur Erzeugung eines gerichteten Gasstrahls
DE3619886C2 (fr)
DE112009001895B4 (de) Ionengatter eines Ionenmobilitätsspektrometers mit dualer Polarität und Verfahren dafür
EP0503748B1 (fr) Procédé pour générer des ions, en particulier pour un spectromètre de masse tel qu'un spectromètre à temps de vol, à partir de molécules thermiquement instables, non-volatiles et de masse élevée
DE102006015535A1 (de) Verfahren und Vorrichtung zur Analyse von Isotopenverhältnissen
DE4303027C2 (de) Massenspektrometer
DE112014001615T5 (de) Vorrichtung und Verfahren für die Flüssigkeitsprobeneinführung
DE69029517T2 (de) Vorrichtung und verfahren zur probenentnahme
EP1915770B1 (fr) Procede et dispositif pour la detection de composes par spectrometrie de masse
WO2005057206A1 (fr) Dispositif pour preparer un echantillon
WO2006064048A1 (fr) Procede et systeme d'etude destines a l'analyse de masse a debit eleve
DE10241545A1 (de) Vorrichtung zur Überführung eines kontinuierlichen Flüssigkeitsstroms in einen Strom aus Flüssigkeitströpfchen
DE102005032983B4 (de) Düsenanordnung
DE4427314C2 (de) Vorrichtung zur Untersuchung von Behältern auf Fremdgase
DE3876803T2 (de) Verfahren und vorrichtung zur elektrothermischen atomisierung von proben.
DE19548977C1 (de) NMR-Meßzelle und Verfahren zur schnellen Mischung mindestens zweier Reaktionsfluide in der NMR-Meßzelle
DE102005005333B4 (de) Verfahren zur Probennahme und Aerosol-Analyse
EP0458010A1 (fr) Procédé et dispositif pour l'analyse de specimens au moyen de spectroscopie par absorption atomique
DE102005000840A1 (de) Verfahren und Vorrichtung zur Elementanalyse mittels Laser-Emissionsspektrometrie
EP1163505A1 (fr) Dispositif pour analyser des elements contenus dans des echantillons de liquides sous forme de gouttelettes
DE3132638C2 (fr)
DE4021239C2 (de) Verfahren und Vorrichtung zum Extrahieren gelöster flüchtiger Substanzen aus Flüssigkeiten in die Gasphase
DE1934487A1 (de) Probengeber fuer Analysengeraete
DE10260071A1 (de) Vorrichtung zur Analyse von in einer Flüssigkeit enthaltenen biologischen Zellen
DE69011509T2 (de) Erzeuger von monodispergiertem Aerosol zur Verwendung mit infraroter Spektrometrie.

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR LV MK YU

RIN1 Information on inventor provided before grant (corrected)

Inventor name: ABEL, BERND

Inventor name: CHARVAT, ALES

Inventor name: TROE, JUERGEN

17P Request for examination filed

Effective date: 20061004

17Q First examination report despatched

Effective date: 20061219

AKX Designation fees paid

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU MC NL PL PT RO SE SI SK TR

RAP3 Party data changed (applicant data changed or rights of an application transferred)

Owner name: GEORG-AUGUST-UNIVERSITAET GOETTINGEN

Owner name: MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENS

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20160227