CN103105352A - Device and method for rapidly detecting surviving unicellular organisms in ship ballast water - Google Patents

Device and method for rapidly detecting surviving unicellular organisms in ship ballast water Download PDF

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CN103105352A
CN103105352A CN2013100326711A CN201310032671A CN103105352A CN 103105352 A CN103105352 A CN 103105352A CN 2013100326711 A CN2013100326711 A CN 2013100326711A CN 201310032671 A CN201310032671 A CN 201310032671A CN 103105352 A CN103105352 A CN 103105352A
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liquid storage
storage hole
channel
ballast
fluidic chip
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孙野青
宋永欣
彭冉
潘新祥
李冬青
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Dalian Maritime University
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Dalian Maritime University
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Abstract

The invention discloses a device and a method for rapidly detecting surviving unicellular organisms in ship ballast water. The device comprises a PDMS micro-fluidic chip and glass substrate G, wherein the PDMS micro-fluidic chip comprises a liquid storage hole A, a liquid storage hole B, a liquid storage hole C, a liquid storage hole D, a liquid storage hole E, a main channel, a first focusing channel, a second focusing channel, a detection channel, a first sorting channel and a second sorting channel, wherein the liquid storage hole A, the liquid storage hole B, the liquid storage hole C, the liquid storage hole D and the liquid storage hole E are inserted into a platinum electrode. The device has the beneficial effects that (1) full-automatic detection, sorting, counting and size determination of the surviving unicellular organisms in the ship ballast water are realized on the micro-fluidic chip, and the device is small in size and is portably and rapidly used for on-site analysis; and (2) a differential micro-fluidic chip and a signal differential amplification mechanism are adopted, and the signal to noise ratio of a detection signal can be improved, so that the detection precision is improved.

Description

The monadic apparatus and method of survival in a kind of fast detecting ballast water for ship
Technical field
The present invention relates to the monadic apparatus and method of survival in a kind of fast detecting ballast water for ship, relate in particular to a kind of apparatus and method that realize the monadic full-automatic detection of survival, sorting, counting and size discrimination in ballast water for ship on micro-fluidic chip.
Background technology
Ballast water for ship is used to drinking water, the trim that boats and ships provide stability and adjust boats and ships, to satisfy the good maneuvering performance requirement of boats and ships.The loading station Ballast Water can guarantee that boats and ships are at zero load or partial unloading and the navigation safety under the severe sea condition condition.
Contain a large amount of hydrobionts in ballast water for ship, and by the external marine biological invasion that ballast water for ship causes, confirmed as four one of threats greatly of harm ocean by Global Environment Facility's tissue (GEF).For better managing and control ballast water for ship, International Maritime Organization (IMO) (IMO) has passed through " Management of Ships ' Ballast Water and sediment management and control pact " (hereinafter to be referred as pact) in 2004, must be through processing before pact mandatory provision ballast water for ship discharging, and in the ballast water for ship after processing: (1) minimum dimension more than or equal to the living body biological number of 50 μ m less than 10/m 3(2) minimum dimension more than or equal to 10 μ m but less than the living body biological number of 50 μ m less than 10/mL; (3) concentration of cholera bacteria (O1 and O139) is less than 1CFU/100mL or 1CFU/g animal plankton sample; (4) colibacillary concentration is less than 250CFU/100mL; (5) concentration of enterococcus is less than 100CFU/100mL.Pact is also stipulated: Port State duly authorized official has the right according to Guidelines see and whether satisfy cleanup standard, but the sample analysis time can not to be caused delay improperly to boats and ships the sampling of taking a sample of the ballast water for ship after processing.
In case pact comes into force, along with and a huge challenge coming is the accordance inspection how Port State carries out relevant ballast water treatment performance.Comprehensive present technology, present microorganism detects and method of counting mainly contains methods such as cultivating counting method, digital image analysis method, the anti-method of solution resistance, cinephotomicrography method, magic lantern mensuration, optical microscope counting method, fluorescent microscope counting method, most probable rate number-PCR counting method (MPN-PCR method), turbidity counting method, flow cytometry, molecular detecting method and biochemical vigor detection.But these methods all need to utilize laboratory relatively ripe equipment and instrument to analyze, and are bulky, time-consuming, can easily not take the scene to, and some detection method can't be distinguished life or death and the size of microorganism.And some detecting instrument needs special technician's operation, therefore is not suitable for.
As seen; the microminiaturization of the maritime affair law enforcement inspection of development under the BWM Convention and portability analytical instrument and equipment have important and urgent research and development demand, and improve China's maritime affair law enforcement level, ensure shipping and the aspect such as protect the marine environment also significant.
Summary of the invention
Be applied to ballast water for ship detection above shortcomings part for solving existing micro-organism test apparatus, the present invention proposes to adopt difference Ku Erte detection of electrons and laser-Induced Fluorescence Detection combination method, by the driven by electroosmosis sample flow, realize on micro-fluidic chip the monadic full-automatic detection of survival, sorting, counting and size discrimination in ballast water for ship.Because sample adopts motorized motions and controls, avoided the use of pump valve, can be integrated into easily the detecting instrument of portability.
The invention provides the monadic apparatus and method of survival in a kind of fast detecting ballast water for ship.
in a kind of fast detecting ballast water for ship, the monadic device of survival, is characterized in that, comprises the PDMS micro-fluidic chip, is positioned at the glass negative G of PDMS micro-fluidic chip below and the sealing-in of PDMS micro-fluidic chip, described PDMS micro-fluidic chip comprises liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E, the main channel, the first focus channel, the second focus channel, sense channel, the first sort channel, the second sort channel, liquid storage hole A is connected with the main channel, liquid storage hole B is connected with the first focus channel, liquid storage hole C is connected with the second focus channel, liquid storage hole D is connected with the first sort channel, liquid storage hole E is connected with the second sort channel, the first sort channel, the second sort channel is connected with the main channel by sense channel, the first focus channel, the second focus channel is connected with the main channel, liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, insert platinum electrode in liquid storage hole E, the other end of the platinum electrode in liquid storage hole A is connected with the positive pole of direct supply, the other end of the platinum electrode in liquid storage hole E passes through resistance R, dpdt relay and potentiometer are connected to the negative pole of direct supply, the other end of the platinum electrode of liquid storage hole D is connected to the negative pole of direct supply by dpdt relay and potentiometer, liquid storage hole B directly is connected with the negative pole of direct supply with platinum electrode in liquid storage hole C, the resistance R two ends are connected to two input ends of differential amplifier circuit by wire, the output terminal of differential amplifier circuit is connected to the input end of ARM, the top of sense channel arranges laser emission probe, the laser of laser emission probe emission enters sense channel by the first optical filter, position facing to sense channel below glass negative G arranges the second optical filter, below the second optical filter, avalanche probe is set, the output terminal of avalanche probe connects the input end of amplifying circuit, and the output terminal of amplifying circuit is connected to ARM.
A kind of employing device claimed in claim 1 carries out the monadic method of survival in the fast detecting ballast water for ship, it is characterized in that, comprises the steps:
1) sample extraction step: extract the ballast for cruising water sample from ballast water for ship to be detected;
2) ballast for cruising water sample and damping fluid drip step: will adopt the ballast for cruising water sample after the FDA dyeing to join in liquid storage hole A; And drip respectively damping fluid in liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E;
3) connecting direct supply carries out electric osmose to the ballast for cruising water sample in liquid storage hole A and transports;
4) when not surviving unicellular organism in the ballast for cruising water sample, the unicellular organism sample all is delivered to liquid storage hole D by the first sort channel under the effect of electroosmotic flow;
5) when having the survival unicellular organism in the ballast for cruising water sample, when the unicellular organism that lives passes through sense channel, the laser that laser emission probe sends excites the unicellular organism with fluorescence through the FDA dyeing, the fluorescence signal that inspires enters avalanche probe through the first optical filter and the second optical filter and carries out the photosignal conversion, after amplifying, signal amplification circuit is sent to ARM from the electric signal of avalanche probe output, and be sent to dpdt relay and make the dpdt relay action, the voltage of liquid storage hole D and liquid storage hole E is exchanged, with or the sorting of unicellular organism sample to hydraulic pressure hole E, finish the action of dpdt relay after lasting Preset Time, make the power up in liquid storage hole D and liquid storage hole E extremely exchange front numerical value,
6) obtaining the detected value of the monadic number of survival and the monadic size of surviving. the monadic number detected value of surviving equals the number of fluorescent pulse signal; The amplitude of the voltage pulse signal that detects according to resistance R two ends is judged the monadic size of survival.
Implement the monadic apparatus and method of survival in fast detecting ballast water for ship of the present invention, have following beneficial effect: (1) adopts Ku Erte detection of electrons and laser-Induced Fluorescence Detection combination method, by the driven by electroosmosis sample flow, realize adopting a small amount of sample and reagent can obtain fast testing result to the monadic full-automatic detection of survival, sorting, counting and size discrimination in ballast water for ship on micro-fluidic chip; (2) adopt micro-fluidic chip to carry out sample detection, and adopt the differential amplifier circuit based on AD620, and mini laser-induced fluorescence detection system, the volume of whole device is very little, can be portable be used to carrying out on-the site analysis.
Description of drawings
Fig. 1 is the structural representation of an embodiment of survival unicellular organism device in fast detecting ballast water for ship of the present invention;
Fig. 2 is the structural representation of an embodiment of micro-fluid chip of the present invention.
In figure: 1, direct supply; 2, potentiometer; 3, dpdt relay; 4, resistance R; 5, differential amplifier circuit; 6, laser emission probe; 7, the first optical filter; 8, the second optical filter; 9, avalanche probe; 10, amplifying circuit; 11, ARM; 12, main channel; 13, the first focus channel; 14, the second focus channel; 15, sense channel; 16, the first sort channel; 17, the second sort channel.
Embodiment
The invention provides the monadic apparatus and method of survival in a kind of fast detecting ballast water for ship, below in conjunction with accompanying drawing, technical scheme of the present invention is elaborated.
Fig. 1 is the structural representation of an embodiment of survival unicellular organism device in fast detecting ballast water for ship of the present invention; Fig. 2 is the structural representation of an embodiment of micro-fluid chip of the present invention, as shown in the figure.
PDMS micro-fluidic chip P comprises liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E, main channel 12, the first focus channel 13, the second focus channel 14, sense channel 15, the first sort channel 16, the second sort channel 17, liquid storage hole A is connected with main channel 12, liquid storage hole B is connected with the first focus channel 13, liquid storage hole C is connected with the second focus channel 14, liquid storage hole D is connected with the first sort channel 16, liquid storage hole E is connected with the second sort channel 17, the first sort channel 16, the second sort channel 17 is connected with main channel 12 by sense channel 15, the first focus channel 13, the second focus channel 14 is connected with main channel 12, liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, insert platinum electrode in liquid storage hole E, the other end of the platinum electrode in liquid storage hole A is connected with the positive pole of direct supply, the other end of the platinum electrode in liquid storage hole E is by resistance R 4, dpdt relay 3 and potentiometer 2 are connected to the negative pole of direct supply 1, the other end of the platinum electrode of liquid storage hole D is connected to the negative pole of direct supply 1 by dpdt relay 3 and potentiometer 2, liquid storage hole B directly is connected with the negative pole of direct supply 1 with platinum electrode in liquid storage hole C, resistance R 4 two ends are connected to two input ends of differential amplifier circuit 5 by wire, the output terminal of differential amplifier circuit 5 is connected to the input end of ARM11.Glass negative G is positioned at below the PDMS micro-fluidic chip, and is integral with PDMS micro-fluidic chip sealing-in sealing-in.
In an embodiment of the present invention, employing is based on the differential amplifier circuit 5 of AD620.
the method for making of PDMS micro-fluidic chip P is: adopt soft lithography soft-lithography to process required PDMS micro-fluidic chip P, namely by printing mask, use the negative photoresist of SU-8, process the anode membrane with main channel and mixed liquor injection channel on silicon-based wafer by uv-exposure, then at wafer upper polydimethylsiloxane, adopt the constant-temperature vacuum stove, solidify under 70~80 ℃ of conditions and obtained to have liquid storage hole A in 3~4 hours, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E, main channel 12, the first focus channel 13, the second focus channel 14, sense channel 15, the first sort channel 16, the PDMS micro-fluidic chip P of the second sort channel 17.
in the fast detecting ballast water for ship, survival unicellular organism device comprises PDMS micro-fluidic chip P, be positioned at the glass negative G of PDMS micro-fluidic chip P below and the sealing-in of PDMS micro-fluidic chip, the liquid storage hole A of PDMS micro-fluidic chip P is connected with main channel 12, the liquid storage hole B of PDMS micro-fluidic chip P is connected with the first focus channel 13, the liquid storage hole C of PDMS micro-fluidic chip P is connected with the second focus channel 14, the liquid storage hole D of PDMS micro-fluidic chip P is connected with the first sort channel 16, the liquid storage hole E of PDMS micro-fluidic chip P is connected with the second sort channel 17, the first sort channel 16, the second sort channel 17 is connected with main channel 12 by sense channel 15, the first focus channel 13, the second focus channel 14 is connected with main channel 12, liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, insert platinum electrode in liquid storage hole E, the other end of the platinum electrode in liquid storage hole A is connected with the positive pole of direct supply, the other end of the platinum electrode in liquid storage hole E is by resistance R 4, dpdt relay 3 and potentiometer 2 are connected to the negative pole of direct supply 1, the other end of the platinum electrode of liquid storage hole D is connected to the negative pole of direct supply 1 by dpdt relay 3 and potentiometer 2, liquid storage hole B directly is connected with the negative pole of direct supply 1 with platinum electrode in liquid storage hole C, resistance R 4 two ends are connected to two input ends of differential amplifier circuit 5 by wire, the output terminal of differential amplifier circuit 5 is connected to the input end of ARM11, the top of sense channel 15 arranges laser emission probe 6, the laser of laser emission probe 6 emissions enters sense channel 5 by the first optical filter 7, position facing to sense channel 15 below glass negative G arranges the second optical filter 8, below the second optical filter 8, avalanche probe 9 is set, the output terminal of avalanche probe 9 connects the input end of amplifying circuit 10, and the output terminal of amplifying circuit 10 is connected to ARM11.
Adopt the unicellular organism device of surviving in the fast detecting ballast water for ship to carry out the monadic method of survival in the fast detecting ballast water for ship, comprise the steps:
1) sample extraction step: extract the ballast for cruising water sample from ballast water for ship to be detected;
2) ballast for cruising water sample and damping fluid drip step: will adopt the ballast for cruising water sample after the FDA dyeing to join in liquid storage hole A; And drip respectively damping fluid in liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E;
3) connecting ballast for cruising water sample in 1 couple of liquid storage hole A of direct supply carries out electric osmose and transports;
4) when not surviving unicellular organism in the ballast for cruising water sample, the unicellular organism sample all is delivered to liquid storage hole D by the first sort channel under the effect of electroosmotic flow;
5) when having the survival unicellular organism in the ballast for cruising water sample, when the unicellular organism that lives passes through sense channel 15, the laser that laser emission probe 6 sends excites the unicellular organism with fluorescence through the FDA dyeing, the fluorescence signal that inspires enters avalanche probe 9 through the first optical filter 7 and the second optical filter 8 and carries out the photosignal conversion, after amplifying, signal amplification circuit 10 is sent to ARM11 from the electric signal of avalanche probe 9 outputs, and be sent to dpdt relay 3 and make dpdt relay 3 actions, the voltage of liquid storage hole D and liquid storage hole E is exchanged, with or the sorting of unicellular organism sample to hydraulic pressure hole E, finish the action of dpdt relay after lasting Preset Time, make the power up in liquid storage hole D and liquid storage hole E extremely exchange front numerical value,
6) obtain the detected value of the monadic number of survival and the monadic size of surviving, the monadic number detected value of surviving equals the number of fluorescent pulse signal; The amplitude of the voltage pulse signal that detects according to resistance two ends is judged the monadic size detection value of survival.When the diameter of the polystyrene standard particle of proofreading and correct use is d MarkThe time, utilize this polystyrene standard particle to test to record the amplitude of the normal voltage pulse signal that resistance two ends detect to be designated as During to the unicellular organism sample test, if the amplitude of the voltage pulse signal that resistance two ends detect is
Figure BDA00002786442000062
According to formula
Figure BDA00002786442000063
Convert, can calculate to obtain the monadic diameter value d of survival 2
Damping fluid of the present invention be for can realize the damping fluid of technical solution of the present invention arbitrarily, for example phosphate buffer or borate buffer solution.As optimal technical scheme, when selecting borate buffer solution to do, the pH value of borate buffer solution is 8-9.
The liquid PDMS that makes PDMS micro-fluidic chip P can be for realizing the material of technical solution of the present invention arbitrarily, for example the Sylgard184 silicones.
ARM11 can be the ARM of the disposable type that can realize technical solution of the present invention, and for example model is the ARM of S3C6410.
The above; only be the better embodiment of the present invention; but protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or changed according to technical scheme of the present invention and inventive concept thereof, within all should being encompassed in protection scope of the present invention.

Claims (2)

1. the monadic device of survival in a fast detecting ballast water for ship, is characterized in that, comprises the PDMS micro-fluidic chip, is positioned at the glass negative G of PDMS micro-fluidic chip below and the sealing-in of PDMS micro-fluidic chip, described PDMS micro-fluidic chip comprises liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E, main channel (12), the first focus channel (13), the second focus channel (14), sense channel (15), the first sort channel (16), the second sort channel (17), liquid storage hole A is connected with main channel (12), liquid storage hole B is connected with the first focus channel (13), liquid storage hole C is connected with the second focus channel (14), liquid storage hole D is connected with the first sort channel (16), liquid storage hole E is connected with the second sort channel (17), the first sort channel (16), the second sort channel (17) is connected with main channel (12) by sense channel (15), the first focus channel (13), the second focus channel (14) is connected with main channel (12), liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, insert platinum electrode in liquid storage hole E, the other end of the platinum electrode in liquid storage hole A is connected with the positive pole of direct supply, the other end of the platinum electrode in liquid storage hole E is by resistance R (4), dpdt relay (3) and potentiometer (2) are connected to the negative pole of direct supply (1), the other end of the platinum electrode of liquid storage hole D is connected to the negative pole of direct supply (1) by dpdt relay (3) and potentiometer (2), liquid storage hole B directly is connected with the negative pole of direct supply (1) with platinum electrode in liquid storage hole C, resistance R (4) two ends are connected to two input ends of differential amplifier circuit (5) by wire, the output terminal of differential amplifier circuit (5) is connected to ARM(11) input end, the top of sense channel (15) arranges laser emission probe (6), the laser of laser emission probe (6) emission enters sense channel (5) by the first optical filter (7), position facing to sense channel (15) below glass negative G arranges the second optical filter (8), in the below of the second optical filter (8), avalanche probe (9) is set, the output terminal of avalanche probe (9) connects the input end of amplifying circuit (10), and the output terminal of amplifying circuit (10) is connected to ARM(11).
2. one kind is adopted device claimed in claim 1 to carry out the monadic method of survival in the fast detecting ballast water for ship, it is characterized in that, comprises the steps:
1) sample extraction step: extract the ballast for cruising water sample from ballast water for ship to be detected;
2) ballast for cruising water sample and damping fluid drip step: will adopt the ballast for cruising water sample after the FDA dyeing to join in liquid storage hole A; And drip respectively damping fluid in liquid storage hole A, liquid storage hole B, liquid storage hole C, liquid storage hole D, liquid storage hole E;
3) connecting direct supply (1) carries out electric osmose to the ballast for cruising water sample in liquid storage hole A and transports;
4) when not surviving unicellular organism in the ballast for cruising water sample, the unicellular organism sample all is delivered to liquid storage hole D by the first sort channel under the effect of electroosmotic flow;
5) when having the survival unicellular organism in the ballast for cruising water sample, when the unicellular organism that lives passes through sense channel (15), the laser that laser emission probe (6) sends excites the unicellular organism with fluorescence through the FDA dyeing, the fluorescence signal that inspires enters avalanche probe (9) through the first optical filter (7) and the second optical filter (8) and carries out the photosignal conversion, after amplifying, signal amplification circuit (10) is sent to ARM(11 from the electric signal of avalanche probe (9) output), and be sent to dpdt relay (3) and make dpdt relay (3) action, the voltage of liquid storage hole D and liquid storage hole E is exchanged, with or the sorting of unicellular organism sample to hydraulic pressure hole E, finish the action of dpdt relay after lasting Preset Time, make the power up in liquid storage hole D and liquid storage hole E extremely exchange front numerical value,
6) obtain the detected value of the monadic number of survival and the monadic size of surviving, the monadic number detected value of surviving equals the number of fluorescent pulse signal; The amplitude of the voltage pulse signal that detects according to resistance R (4) two ends is judged the monadic size of survival.
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