C4Dマイクロチップ電気泳動の原理および使用方法

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Video 1. C4Dを使ったマイクロチップ電位泳動法の手順

非接触電導度検出器(C4D)を用いたマイクロチップ電気泳動法(MCE)の手順

はじめに

図 1. マイクロチップ

このアプリケーションノートでは、静電結合型非接触電気 電導度検出器(C4D)を使ったマイクロチップ電気泳動法(MCE-C4D)の操作手順をステップ-バイ-ステップで説明します。 eDAQ マイクロチップ電気泳動システムには、C4D データシステム、C4D マイクロチッププラットフォーム、高電圧シーケンサー、C4D の電極を埋め込んだマイクロチップ、カチオン及びアニオンを含む標準液キットが含まれています。

図 1 は使用するマイクロチップの構造を示した写真です。

必要な装置

ER255 及び ER455 マイクロチップ電気泳動システム

フローティングインジェクション

ここではフローティングインジェクション法によるマイクロチップ電気泳動の操作手順を紹介します。この種のインジェクションでは、チップにある4つのリザーバの内、2箇所に電圧を設定します。従ってこの方法では、1台の高電圧シーケンサを使います。 フローティングインジェクションを行う間は、チップの両端(リザーバ 1 と 3 の間)に 1000 V かけます。チップのチャネル(流路)はダブル-T字の構造で、この電位がチャネルの僅かな間隙全体に及びます。分離工程ではチップに沿って(リザーバ2と4) 1000 V かかることになります。この結果、サンプルは検出器とリザーバ4の方に移動し、それにつれてイオンが分離します。 ゲートインジェクション法では後述するように、4か所全てのリザーバで電圧のコントローラが必要(従って2台の高電圧シーケンサーが必要)となります。

必要な装置

表 1. チッププラットフォームと HVS を接続し、フローティングインジェクションを行う。

eDAQ マイクロチップ電気泳動キットには多くのコンポーネントとケーブルが含まれています。次の手順に従ってください。

  1. まず、パッキングリストに記載した品目が全て揃っているか確認してください。
  2. コンピュータに Sequencer ソフトウェアと PowerChrom ソフトウェアをインストールします。インストールしたバージョンが最新バージョンなのか edaq.com/software_dnloads.html で確認してください。
  3. 操作マニュアルに従って、高電圧シーケンサ(HVS)をUSB ケーブルでコンピュータに接続します。但し電源は未だ入れないでください
  4. 同様に、C4Dデータシステムをマニュアルに従いUSBケーブルでコンピュータにつなぎます。電源は入れない事。
  5. HDMI ケーブルを使って、チッププラットフォームをC4Dデータシステムにつなぎます。
  6. 表1に従って、カラーで識別した高電圧ケーブルを使ってチッププラットフォームをHVSに接続します。
    • 黒色の HVS ケーブルは2本あるますので必ず正しい方をつないでください。45 mm マイクロチップを使っている場合は、プラットフォーム中央部の黒色ケーブルを使います。
    • チッププラットフォームと HVS 間はインターロックケーブルで接続します。注:カバープレートがプラットフォームから持ち上がっている場合は、インターロックがかかり電源は入りません。絶対にインターロックケーブルを迂回させないこと。
  7. チップホルダーは必ずアースを取り、シグナルのノイズを抑えます。緑のグランドケーブルを使ってチッププラットフォームのグランドコネクターと C4D データシステム後部の緑のコネクターとをつなぎます。同様に、もう一本のグランドケーブルで C4D システムの緑のコネクターと HVS 後部の緑のコネクターとをつなぎます。
    Video 2. HV シーケンサーからPowerChromソフトウェアをトリガーする設置法
  8. HVS からPowerChrom ソフトウェアにトリガーを掛けて記録を開始させることができます。赤と黒のトリガーケーブルを使って、HVS 後部の “CTL1 +” と C4D データシステムの “TRIG +” とをつなぎ、HVS の “CTL1 –” と C4D の “TRIG –” をつなぎます。 Video 2 参照。
  9. HVS の電源を入れます。初めて使う場合はドライバーがインストールされます。
  10. Sequencer ソフトウェアを開き、 and ensure you are able to connect to the HVS by clicking on the Online button at the top of the screen. You may need to go into the File, Preferences menu to select the correct Connection Serial Port for the HVS. You can use the Serial Port Monitor software (from the eDAQ website) to see which COM port the HVS is connected to. Move and resize the Sequencer software screen so it occupies the left half of the screen.
  11. Turn on the C4D Data System. The driver will be installed if this is the first use.
  12. Open the PowerChrom software. Ensure the software has setup the hardware unit; you should see the Easy Access window, not the Hardware Unavailable window. Move and resize the PowerChrom software screen so it occupies the right half of the screen.
  13. Setup the trigger commands in the software packages as shown below:
    • Sequencer software: in the menu File, Preferences, select Contact Closure.
    • PowerChrom software: in the menu Edit, Preferences, Digital IO Settings, select External Trigger Mode as Voltage Level (TTL).
    • PowerChrom software: in the Manual Sampling window, Inject Settings, select Wait for Inject.
    • Sequencer software: in the later step, when you are setting up the sequence, remember to select High/Closed, under Digital Output 1, during the separation step, as this will send the trigger command. Also remember to select Low/Open in the very last step (after the separation is complete).
  14. Use gloves to place the empty microchip in the Chip Platform. Ensure that the four circular C4D connectors lie on top of the four pins on the platform. Figure 2 shows a microchip in the platform.
  15. Place the reservoir cover on.
    • Ensure that the O-rings underneath the cover are in place and are dry; wipe with a tissue if necessary.
    • Don’t turn the screws too tight; the screws should be turned finger-tight.
    • Ensure that the reservoir cover is sitting flat and level with the base of the platform.
  16. Place the cover plate on top of the platform. You may need to move the high voltage cables slightly to ensure it is sitting flat and level with the base of the platform

Sequencer Software

  1. Ensure the Sequencer software is open and in Online mode.
  2. Setup a sequence as shown in either Table 2 for separating cations, or Table 3 for separating anions. These use a Floating Injection.


Table 2. Sequence for separating cations using a Floating Injection
Table 3. Sequence for separating anions using a Floating Injection

PowerChrom Software and Pipetting Solutions

  1. Ensure the PowerChrom software is open.
  2. In the Easy Access window, click Manual Run.
  3. In the Manual Sampling window, click Inject Settings and select Wait for Inject (Start recording immediately) and OK.
  4. In the Manual Sampling window, enter Stop sampling 2.00 min after Inject.
  5. Click Hardware Settings. Enter the following settings and then OK:
    • Sampling speed = 100/s
    • Channel 1: Input = Input 1, Range = 50 mV
    • Channel 2: Input = Off
  6. Click C4D Amplifier and enter the following settings:
    • Low Pass = 5 Hz
    • In Offset, click Zero
  7. Dilute the sample from 1 mM to 100 µM with deionised water.
    Figure 3. Pipetting solutions into the microchip reservoirs
  8. Pipette 50 µL of the BGE into Reservoir 4. It should take less than one second for capillary action to fill the channel with the solution. This can be confirmed by observing a change in conductivity in the C4D signal.
    • Keep the pipette vertical while pipetting solutions into the reservoirs.
    • Make sure that the pipette tip gently touches the sloping walls of the reservoir, as you deliver the solution. This will prevent the formation of an air bubble at the bottom of the reservoir.
    • Use different pipette tips when transferring the BGE and the sample to the reservoirs, and when removing solutions from the reservoirs. This will avoid contaminating the solutions.
  9. In the PowerChrom software:
    • If you already know the C4D settings for Frequency, Amplitude and Headstage Gain, then enter these values now in the C4D Amplifier window, or
    • If you don’t know the C4D settings, use the C4D Profiler to optimise the C4D settings, as described in a separate application note. This must be done with BGE in the channel.
  10. If separating cations, pipette 50 µL of the BGE into Reservoir 2 and Reservoir 3. Pipette 50 µL of the sample into Reservoir 1.
  11. If separating anions, pipette 50 µL of the BGE into Reservoir 2. Pipette 50 µL of the sample into Reservoir 1 and Reservoir 3.
  12. Visually check that you have the same volume of solution in each of the reservoirs.
  13. Place the cover plate onto the holder.
  14. Click Zero in the Offset box.
  15. You may need to select a smaller value for the Range at this point. Click OK twice to return to the Manual Sampling window.
  16. Arm the HVS by pressing and holding the red button on the front of the HVS unit.
  17. You are now ready to analyse your sample. In the PowerChrom software, click “Start”.
  18. In the Sequencer software, click “Run” to start the HVS sequence. This should trigger the PowerChrom software to start recording.

Results for Cations

The electropherogram for the cations, run at 100 µM concentration, is shown in Figure 4. As predicted by Peakmaster software, three negative peaks are observed. The peaks are negative because the three cations Li+, Na+ and K+ are less conductive than the H+ cation they are displacing in the background electrolyte. The electropherogram can be recorded with positive peaks by selecting Invert in the C4D Amplifier window, in the Hardware Settings of the PowerChrom software.

Figure 4 shows the data with a sample injection time of 20 seconds. Increasing the injection time, to 30 and 40 seconds, resulted in larger peaks but the separation of the peaks was not as good.

Figure 4. Cations at 100 µM concentration

Results for Anions

Figure 5 shows the electropherogram for anion analysed at 1mM. This concentration will overload the system, resulting in one large peak which cannot be resolved.

The solution for running anions should be diluted to 100 µM with deionised water. The electropherogram is shown in Figure 6. Peakmaster software predicts the two positive peaks obtained, where the HSO4- and NO3- peaks cannot be resolved. Separating the anions under these conditions will not produce an EOF peak, because the EOF travels away from the detector, towards Reservoir 2.

Figure 5. Anions overloading at 1 mM concentration
Figure 6. Anions at 100 µM concentration

Procedure for Changing the Sample

  1. Disarm the HVS.
  2. Remove the cover plate from the platform.
  3. Remove the sample from the sample reservoir using a pipette.
  4. Flush the sample reservoir a few times using deionised water.
  5. Pipette 50 µL of the new sample into the sample reservoir.
  6. Replace the cover plate.
  7. Arm the HVS.
  8. Begin the analysis of the new sample.

Procedure for Removing the Microchip

  1. Disarm the HVS.
  2. Remove the cover plate from the platform.
  3. Remove the solution from each of the reservoirs using a pipette.
  4. To prevent formation of salt crystals inside the channel during storage, pipette deionised water into each of the reservoirs.
  5. Flush the microchip by placing the syringe into the Reservoir 4 and gently pressing on the syringe, holding the pressure for ten seconds.
  6. Remove the deionised water from each of the reservoirs using a pipette.
  7. Remove the reservoir cover and dry the o-rings underneath the cover using a tissue.
  8. Remove the chip.

Notes

It is sometimes necessary to filter the BGE and sample solutions to prevent particles from blocking the narrow channel of the chip. This can be achieved by using a filter which fits onto the end of the syringe.

It may also be necessary to place the BGE and sample solutions in an ultra-sonic bath to remove any dissolved air in the solutions. Dissolved air can cause the formation of air bubbles in the channel, which can result in an electric arc when the high voltage is applied. This produces high temperatures in the channel which can damage the channel of the chip.

Observing the current flowing through the channel of the chip can be useful when developing a method. This current is displayed in the Sequencer software, and it can be recorded from the monitor connectors at the back of the HVS. If the current reads zero, or is very noisy, during the separation step, this suggests there is an air bubble in channel.

If the channel of the chip becomes blocked with particles, it may be possible to clear the obstruction, either by injecting air into one end of the channel using a syringe, or by using a weak vacuum at one end of the channel. Use a lint-free tissues, as opposed to normal tissues, to prevent particles from blocking the channel of the chip.

If the sample peaks get progressively smaller over successive runs, this may be due to the depletion of ions in the sample reservoir close to the start of the channel. You can mix the sample in the reservoir, by using a pipette to suck the sample in and out of the pipette a few times.

If the baseline begins to drift a lot during the analysis, it may be useful to flush the chip using the syringe. Place the syringe into the outlet reservoir and gently press on the syringe and hold the pressure for ten seconds.

You may have to the alter the Offset and change the Range several times during a series of runs, but do NOT alter the Frequency, Amplitude or Headstage Gain settings until you have completed all the calibration and sample runs in an experiment.

Cleaning and Storing the Chip

The chip can be rinsed and emptied while it is still in the Chip Platform. This is done by pushing the nozzle of a syringe into the sloping reservoir holes of the reservoir cover and gently pushing on the plunger to apply pressure.

Alternatively, this can be done with the chip removed from the Chip Platform, by using a syringe with a rubber O-ring connected to its nozzle. To make this device, cut most of the nozzle from a syringe and glue a rubber O-ring to the end of the syringe. The O-ring is then pushed down onto the glass surface of the chip above the reservoir. The plunger is gently pushed to force liquid or air through the channel of the chip.

For the optimum performance, the chip should be properly treated after use to clean the glass surface and restore the EOF properties. Please follow the instructions provided by the chip manufacturer.

Gated Injection

Introduction

The previous experiment described a Floating Injection, where the reservoirs at the ends of the long separating channel (Reservoirs 2 and 4) are disconnected from the High Voltage Sequencer during the sample injection step. An alternative type of injection, called a Gated Injection, may also be used. A Gated Injection requires two units of the ER230 High Voltage Sequencer, as the voltages at all four reservoirs need to be controlled.

Procedure

Table 4. Connecting Chip Platform to HVS for a Gated Injection

The hardware should be setup as described for the Floating Injection, except the Chip Platform should be connected to the HVS as shown in Table 4.

In the Sequencer software, setup a sequence as shown in Table 5 for cations. For the separation of anions, the polarities of the applied voltages are reverse (set all the voltages in Table 5 to negative values).

In the sequence, make sure that you enter the trigger command for the correct HVS. For example, if you have connected the trigger cable to the Master HVS hardware, make sure you enter the “High/Closed” command in the Digital Out column for the Master HVS.

The sample should be diluted to 100 µM with deionised water. Pipette 50 µL of BGE into Reservoir 4, 3 and 1, and 50 µL of sample into Reservoir 2.

Follow the rest of the instructions to start the PowerChrom and Sequencer softwares.

Table 5. Sequence for separating cations using a Gated Injection
Figure 7. Flow during Initial/Separation Step
Figure 8. Flow during Injection Step

Flow of Sample and BGE

During the initial/separation step (shown in Figure 7) there is a constant flow of sample from Reservoir 2 to Reservoir 1, while the BGE flows from Reservoir 3 to Reservoir 4. There is also flow of BGE from Reservoir 3 to Reservoir 1, which keeps the sample out of the separation channel.

After 45 seconds, the injection step occurs (shown in Figure 8). The voltage at Reservoir 3 is reduced, from 1000V to 4000V, for a fraction of a second. This allows a plug of sample into the separation channel.

When the voltage at Reservoir 3 is restored to its original value, the sample plug is broken off and separated as it flows towards Reservoir 4 and the detector.

Notes for Gated Injections

The amount of sample injected is a function of the length of time of the injection. The formation of the sample plug injected into the separation channel is a combination of electromigration and EOF (if the EOF is in the same direction as the electromigration). This type of injection may produce an electrokinetic bias: faster migrating compounds are introduced into the separation channel in a greater quantity than slower migrating compounds.

Contamination in the chip can reduce the surface charge on the surface of the channel. This explains why the EOF peak may be smaller, or may not be seen at all, when using a chip had been used many times.