Micro-Spectrohpotometer USERS MANUAL

Transcription

1 Nano-100 Micro-Spectrohpotometer USERS MANUAL Version 1.0 HANGZHOU ALLSHENG INSTRUMENTS CO., LTD. Foreword Thank you for purchasing our Products: Micro-Spectrohpotometer. This

2 Manual for users contains function and operation of the Instrument. In order to use the instrument properly, please read this manual carefully before using the Instrument. Keep it for later use when you meet with difficulties. Opening Check Please check the Instrument and Appendix with the packing list when you first open the instrument packing case. If you find there is something wrong with the Instrument and the Appendix, do contact the vendor or the producer. HANGZHOU ALLSHENG INSTRUMENTS CO., LTD. ADD: No.2, Xiyuan Road 6th, Economic Park of Science and Technology of the Westlake, Town of Sandun, Hangzhou, China Tel.: Fax: Website: File No.:AS64SM Version No.:Version 1 st, Nov. 2011

3 Safety Warnings and Guidelines 1 Important operation information of the security: Before the users operation, they should have a perfect conception of how to use the Instrument. Therefore, read this Manual carefully before using it. Operation before reading the Manual is forbidden. Read the guidelines and directions below and carry out the countermeasure according to them. 2 Security: The operation, maintenance and repair of the Instrument should comply with the basic guidelines and the remarked warning below. If you don t comply with them, it will have effect on the scheduled using life of the Instrument and the protection provided. This product is indoor Instrument. Read the Manual carefully before operation, or it may cause injured. The expert of wiring equipment can operate this Instrument. The operator should not open or repair the Instrument by himself, which will result in losing the qualification of repair guarantee or occur accident. If there is some wrong with the Instrument, the company will repair it. Power off when you finish your work. Pull off the connector plug when there s long time no use of the Instrument and cover it with a cloth or plastic paper to prevent from dust. Pull the connector plug from the jack at once in the following case, and contact the vendor: There is some liquid flowing into the Instrument; Drenched or fire burned. Abnormal operation: such as abnormal sound or smell. Instrument dropping or outer shell damaged. The function has obviously changed.

4 3 The maintenance of Instrument The pedestal should be cleaned by the cloth stained with alcohol. If there are smutches on the Instrument, clean them with cloth stained with alcohol.

5 CONTENTS Chapter 1 Introduction Chapter 2 Specifications The normal operating condition The basic parameters and performance Chapter 3 Test Principle Chapter 4 Initial Setup Chapter 5 Software Operation

6 Chapter 1 Introduction Chapter 1 Introduction The Nano-100 is a full-spectrum ( nm) spectrophotometer that measures 0.5-2ul samples with high accuracy and reproducibility. It utilizes a patented sample retention technology that employs surface tension alone to hold the sample in place. This eliminates the need for cumbersome cuvettes and other sample containment devices and allows for clean up in seconds. In addition,the Nano-100 has the capability to measure highly concentrated samples without dilution(100x higher concentration than the samples measured by a standard cuvette spectrophotometer). 1

7 Nano-100 manual Chapter 2 Specification Chapter 2 Specifications 1. The normal operating condition Ambient temperature:5 C 35 C The relative humidity: 70% Power Supply:DC24V 2A 2. The basic parameters and performance Model Parameter Nano-100 Minimum Sample Size Path Length Light Source Detector Type Wavelength Range Wavelength Accuracy Spectral Resolution 0.5ul-2ul (2ul advised) 0.2mm or 1mm Xenon flash lamp 3864 element linear silicon CCD array nm ±1 nm 3nm(FWHM@Hg 253.7nm) Absorbance Precision Absorbance Accuracy Absorbance Range Detection limit Maximum Detection Concentration Detection Time Dimension (mm) Net Weight (kg) Pedestal Material Operating Voltage 0.003Abs(1mm path length) 1%(at 0.76 at 350nm) (10mm equivalent) 8ng/ul dsdna 5,000ng/ul(dsDNA) 10-20s mm 2.5 kg SUS304 24V Power 12-18W Software Compatibility Windows XP Vista(32bit)and Win7(32bit) 2

8 Nano-100 manual Chapter 3 Test Principle Chapter 3 Test Principle 1. Samples Retention System A 2ul sample is pipetted onto the end of a fiber optic cable. It is at least to use 0.5ul sample to measure high concentration nucleic acid and protein A280. A second fiber optic cable (the source fiber) is then brought into contact with the liquid sample causing the liquid to bridge the gap between the fiber optic ends. A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array is used to analyze the light after passing through the sample. The instrument is controlled by special software run from a PC, and the data is logged in an Excel file on the PC. 2. Sample Size Requirements Although sample size is not critical, it is essential that the liquid column be formed so that the gap between the upper and lower measurement pedestals is bridged with samples The hydrophobic between the water molecules is the main factor of surface tension. In general, the presence solute of liquids ((including protein, DNA,RNA, salt ion, detergent molecule) can significantly reduce surface tension. Although, for most samples, a 1ul sample size is enough, a 2ul sample size is recommended for protein measurements. Field experience indicates that the following volumes are sufficient to ensure reproducibility: Aqueous solution of nucleic acid:1ul Purified protein:2ul Microbial cell suspension:2ul Others:2ul It is best to use a precision pipettor (0-2ul) with precision tips to assure the 3

9 Nano-100 manual Chapter 3 Test Principle sufficient sample ( 1-2ul) is used. Lower precision pipettors (0-10ul and larger) are not as good as at delivering 1 ul volumes to the measurement pedestal. If you are unsure about your sample characteristics or users or pipettor accuracy, a 2 ul sample is recommended. 3. Basic use 3.1 With the sampling arm open, pipette the sample onto the lower measurement pedestal. 3.2 Close the sampling arm and initiate a spectral measurement using the operating software on the PC. The sample column is automatically drawn between the upper and lower measurement pedestals and the spectral measurement made. 3.3 When the measurement is complete, open the sampling arm and wipe the sample from both the upper and lower pedestals using a soft laboratory wipe. Simple wiping prevents sample carryover in successive measurements for sample. 4. Blanking and Absorbance Calculation When NANO-100 Spectrophotometer is blanked, a spectrum is taken a reference material (blank) and stored in memory as an array of light intensities by wavelength. When a measurement of a sample is taken, the intensity of light that has transmitted 4

10 Nano-100 manual Chapter 3 Test Principle through the sample is recorded. The sample intensities along with the bland intensities are used to calculate the sample absorbance according to the following equation. Thus, the measured light intensity of both the sample and of the blank are required to calculate the absorbance at a given wavelength. The Beer-Lambert equation is used to correlate the calculated absorbance with concentration. A=absorbance represented in absorbance units (A) ε=the wavelength dependent molar absorptivity (unit: L/mol*cm) b=path length (unit: cm) c=sample concentration (unit: mol/l) Contrasted solution, or blank solution,is normally solvent to dissolve the targeting molecular and it need to be same with the samples in PH and ionic strength 5. Blank Cycle For most consistent results, it is best to begin any measurement session with a blanking cycle. This will assure the user that the instrument is working well and that the pedestal is clean. To perform a blanking cycle, perform the following: 1. Load a blank sample onto the lower measurement pedestal and lower the sampling arm into the 'down' position. 2. Click 'Blank' button to make a blank and save the reference spectra. 3. Analyze an aliquot of the blanking solution as though it were a sample. This is done using the 'Measure' button. The result should be a spectrum with a relatively flat baseline, and the absorbance value change should be within 0.04A(10mm path). 4. Wipe the both pedestals and repeat the process until the spectrum is within 0.05A (10mm path). Although, there is no need to make a blanking calculation between every sample, it is best to make a blanking calculation every 30min when measure several samples. After 30min, the time for making last blanking will display in the status bar of the software. 5

11 Chapter 4 Initial Setup Chapter 4 Initial Setup 1. Installation requirement: Computer requirements: Microsoft Windows XP Win7(32bit)or Vista(32bit)operating system. 1.5GHz or higher processor. 1GB or more of RAM(2GB for Vista operation system). 100MB of free hard disk space. Open USB port(the instrument can only be connected via the USB port). Microsoft office Installation preparation: The system software must be loaded onto the PC before the USB cable is connected. Administrator access on the PC is required to install the software. 3. Installation steps: 3.1 Close all programs and make sure that USB cable is unplugged. 3.2 Insert the operation software CD in the CD drive of the PC. Click and then enter into the installation step Step1:Select Setup Language: Step2:Click Next twice and then input the password supplied by the manufacturer (initial password:123456) 6

12 Chapter 4 Initial Setup Click next Step3:Select Destination Location Step4: Select shortcut. 7

13 Chapter 4 Initial Setup Step5:select default setting until the installation ends. Click the Finish button and begin the installation. 3.3 Connect the USB cable to instrument and the computer (installed with software). Turn on the power switch; the red indicator will be bright. Normally, the computer will remind the user to find a new hardware Dialog box. In this time, choose the last item, and click Next to enter into next step. Then select Install from a list or specific location (Advanced). 8

14 Chapter 4 Initial Setup 3.4 Finally, Input Nano 100 Drive folder path, such as C:\Program Files\Nano 100\ Nano 100 Drive in Include this location in the search and then click Next. The drive will start installation. 3.5 After installation, there will be a prompt Dialog box 9

15 Chapter 4 Initial Setup If the computer is prompted to install the driver again, repeat the above process. The computer equipment manager will have the following tips after driver installation is successful. If the Device Manager still display?, it means that this device hasn t been installed properly. Select the device again and click the right mouse button to reinstall the device. Then the user can open the running test software. At the same time, the user can see.net 2.0 frame in the Add or Remove Programs. 10

16 Chapter 5 Software Operation Chapter 5 Software Operation 1. General introduction 1.1 USB cable connection To make measurements with the instrument, connect the USB cable to instrument and the PC, plug in the 24V power supply and connect to the power input at the back of the instrument. The power supply can remain plugged into the Nano-100 while the instrument is not in use. The unit is in standby mode, power consumption is 5W and the flashlamp is not energized. After plugging tin the 24V power supply, utilize a power switch, there will be a red indication in the top of the machine. The green light indicates that the unit is working. 1.2 Software features There are two parts in Nano-100 software, status bar and working key in the left part and display data window in the right part. (1) Taskbar 11

17 Chapter 5 Software Operation The taskbar options include the following options: Home display the application main My Date- restore the sample data saved in the folder specified by the user Diagnostics- test instrument if it is connected properly Options- enter into Debug mode (2) Application selection region Click to select the (color-keyed) type of the sample, and then enter the selected type interface. (3) Function key When the application is opened, the following five function keys are displayed in the top of left column: Blank - Use dissolved sample buffer to make a blank. Before making a sample measurement, a blank must be measured. 12

18 Chapter 5 Software Operation Measure Initiate the measurement for the samples To Excel The detail absorbance data of present measurement is stored in the Excel table which is specified by user. To Picture To store the software (the user is working) interface image Print Print a copy of current data and corresponding spectrum to the default printer. (4)Main menu bar option File The dropdown menu of file menu includes the following options: New - The menu for application operated by specific user group, equivalent to the Home key. Open- Restore the sample data saved in the folder specified by the user, equivalent to My Date key. Save - The detail absorbance data of present measurement is stored in the Excel table which is specified by user. It is equivalent to To Excel Key. Exit Exit program Tools The dropdown menu of tools menu includes the following options: Check correction Test instrument if it is connected properly, it is equivalent to Diagnostics key. Dye Edit Edit dye information. Language - Select operation language. Debug - The dropdown menu of debug menu includes the following options: DebugEnable -Enter or exit the Debuge mode Help The dropdown menu of help menu includes the following options: Contents - Display electronic version of the manual. About- Display information about the software. 2. Applications UV/VIS spectrophotometry is simple for small samples by using Nano-100 Spectrophotometer. The small sample requirement and ease of use make the Nano-100 Spectrophotometer ideally suited for measuring: Nucleic acid concentration and purity of nucleic acid samples up to 5000ng / UL (dsdna) without dilution General UV Vis spectrophotometry Purified protein analysis(a280) Fluorescent dye density of Micro array samples 13

19 Chapter 5 Software Operation 2.1 Quick start (1) Double-click the software icon and select the interested application from the right column (2) Use suitable buffer to create a blank. Load a blank sample onto the lower measurement pedestal and lower the sampling arm into the down position. Then, click on the Blank button. (3) When the measurement is complete, wipe the blanking buffer from both pedestals using a laboratory wipe. Input the sample name in the suitable position and pipette 2ul sample to make measurement. Note: each sample must be pipette freshly After measurement: Wipe the sample from both upper and lower pedestals upon completion of each sample measurement by using clean dust-free paper, so that it can do next sample measurement. The concentration limit values of measurement as below: 14

20 Chapter 5 Software Operation 3. Nucleic Acids measurement 3.1 General Information Nucleic acid samples can be readily checked for concentration and quality using Nano-100 Spectrophotometer. To measure nucleic acid samples select the Nucleic Acid application module. The Beer-Lambert equation is used to calculate nucleic acid concentration. Please see following equation. C=nucleic acid concentration (unit: ng/ml) A=the absorbance in AU ε=the wavelength-dependent extinction coefficient (unit: ng-cm/ml) b=path length (unit: cm) The generally accepted extinction coefficients for nucleic acids are: Double-stranded DNA:50ng-cm/ul Single-stranded DNA:33ng-cm/ul RNA:40ng-cm/ul When choosing pedestal mode, Nano-100 Spectrophotometer can measure the high concentration without dilution by using 1.00mm and 0.2mm short path length to measure. Note: The absorbance value of nucleic acid measurement is consistency of the reading value under 1cm path length. The Nano-100 spectrophotometer can accurately measure double-stranded DNA samples up to 5000ng/ul without dilution. For each sample, software will automatically optimize the best path length to make measurement. When the optical intensity (after measurement sample extinction) is lower than 200(under 1cm path length), software will inform the customer to choose shorter path length to make sure the precision of the measurement. Unique screen is shown as below. 15

21 Chapter 5 Software Operation The data shown in the spectrum image are consistency of the reading value under 10mm path length. The right column of spectrum includes following information: Name-Input the sample name: when sample measurement, the sample name should be input. Type-Used to select the type of nucleic acid being measured. The user can select DNA-50 for dsdna, RNA-40 for RNA, ssdna-33 for single-stranded DNA. ID--Display the serial number for the sample being measured. The user can open the software to start recording. A260-Absorbance of the sample at 260nm with 10mm path A280-Absorbance of the sample at 280nm with 10mm path 260/280-Ratio of sample absorbance at 260nm and 280nm. The ratio of absorbance at 260 and 280nm is used to assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as pure for DNA; a ratio of ~2.0 is generally accepted as pure for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280nm. 260/230-ratio of sample absorbance at 260 and 230nm. This is a secondary measure of nucleic acid purity. The 260/230 values of pure nucleic acid are often higher than the respective 260/280 values. They are commonly in the range of If the ratio is appreciably lower, this may indicate the presence of co-purified contaminants. Baseline correction-if select the baseline correction, the default correction wavelength is 16

22 Chapter 5 Software Operation 340nm. The user can input different wavelength correction according to measurement requirements. In any measurement, the baseline is automatically set to the absorbance value of selectable wavelength. All readings under wavelengths should be minus this value. Note: The user may elect to turn off the baseline correction, which may result in the spectra being offset from the baseline. 3.2 Nucleic Acids concentration application steps 1) In the main menu, select nucleic acid mode. It enter into the Nucleic Acids measurement interface. 2) The users select the type of Nucleic Acid. The user can select 'DNA-50', 'RNA-40', 'ssdna-33' and 'Other'. The default is DNA-50. 3) In the specific location, input sample name and item number. 4) Create a blanking by using suitable solution. Blank solution,is normally solvent to dissolve the targeting molecular and it need to be same with the samples in PH and ionic strength. Load a 1-2ul blank sample onto the lower measurement pedestal and lower the sampling arm into the 'down' position. Click 'Blank' button to make a blank. When the correction is complete, the intensity curve will come out. Wipe the sample from both the upper and lower pedestals using soft laboratory wipe and measure the next solution. 5) Load a 1-2ul standby sample onto the lower measurement pedestal and lower the sampling arm into the 'down' position. When the correction is complete, the absorbance curve will come out. In the meantime, the data for this measurement will show in the measurement result area. The default concentration unit is ng/ul. The default standard wavelength is 340nm. If the users want to choose a new standard wavelength, enter into Debug mode. In the meantime, the measurement results will be added into the measurement result list in the List. If the users want to see more detail absorbance intensity information, they can see them in Show Detail in List. The absorbance intensity data can be stored in Excel file by clicking To Excel. When click My data button to open the file, the measurement result and absorbance curve will be restored in the software. 6) When the measurement is complete, a new laboratory wipe should be used to wipe the pedestals. In this way, the users can do next measurement. If measure the same lot samples, the users do not need to re-blank. It is advisable to do the blanking every 15min at least. 7) The software automatically adds the measurement results into the measurement results list in List. It is best to click 'save' button to save them in the user-specified region before closing the software, since the data will disappear when closing the software. 17

23 Chapter 5 Software Operation Fig 1.Nucleic Acids Measurement Result Fig2. Measurement Results List 4. Protein A General Information 18

24 Chapter 5 Software Operation Proteins, unlike nucleic acids, can exhibit considerable diversity. Protein A280 method is applicable to purified proteins (includes Trp, Tyr residues or Cys-Cys disulfide) exhibiting absorbance at 280nm. It does not require gereration of a standard curve. The software calculate the protein concentration directly after measure the absorbance value. These methods for measure the colors like BCA, Pierce 660nm, Bradford and Lowry, are usually applicable for the samples with uncertain extinction coefficient or cell Iysate. The Protein A280 displays UV spectrum, measures the protein s absorbance at 280nm and calculate the concentration (mg/ml). Like the Nucleic Acide mode, it displays and records 10mm equivalent data. Measurement Concentration Range The Nano-100 Spectrophotometer will accurately measure protein samples up to 100mg/ml BSA) with dilution. When the optical intensity (after measurement sample extinction) is lower than 200(under 1cm path length), software will inform the customer to choose shorter path length to make sure the precision of the measurement. Unique screen is shown as below. The hydrophobic between the water molecules is the main factor of surface tension. In general, the presence solute of liquids ((including protein, DNA,RNA, salt ion, detergent molecule) can significantly reduce surface tension. Although, for most samples, a 1ul sample size is enough, a 2ul sample size is recommended for protein measurements that the liquid column be formed. Screen display: 19

25 Chapter 5 Software Operation The data shown in the spectrum image are consistency of the reading value at 10mm path length. The right column of spectrum includes following information: Name-Input the sample name: when sample measurement, the sample name should be input. Type-Used to select the type of nucleic acid being measured. The user can select A280 for A280, BSA for BSA, lgg for lgg, and 'Lysozyme' for Lysozyme. ID--Display the serial number for the sample being measured. The user can open the software to start recording. A260-Absorbance of the sample at 260nm with 10mm path A280-Absorbance of the sample at 280nm with 10mm path 260/280-Ratio of sample absorbance at 260nm and 280nm. 260/230-ratio of sample absorbance at 260 and 230nm. Baseline correction-if select the baseline correction, the default correction wavelength is 340nm. The users can input different wavelength correction according to measurement requirements. In any measurement, the baseline is readings under wavelengths should be minus this value. Note: The user may elect to turn off the baseline correction, which may result in the spectra being offset from the baseline. 4.2 Protein A280 concentration application steps 1) In the main menu, select Protein A280 mode. It enter into the protein measurement interface. 2) The users select the type of Protein. The user can select 'A280', 'BSA', 'lgg' and 'Lysozyme'. The default is A280. 3) In the specific location, input sample name and item number. 4) Create a blanking by using suitable solution. Blank solution,is normally solvent to dissolve the targeting molecular and it need to be same with the samples in PH and ionic strength. Load a 1-2ul blank sample onto the lower measurement pedestal and lower the sampling arm into the 'down' position. Click 'Blank' button to make a blank. When the correction is complete, the intensity curve will come out. Wipe the sample from both the upper and lower pedestals using soft laboratory wipe and measure the next solution. 5) Load a 1-2ul standby sample onto the lower measurement pedestal and lower the sampling arm into the 'down' position. When the correction is complete, the absorbance curve will come out. In the meantime, the data for this measurement will show in the measurement result area. The default concentration unit is mg/ml. The default standard wavelength is 340nm. If the users want to choose a new standard wavelength, enter into Debug mode. In the meantime, the measurement results will be added into the 20

26 Chapter 5 Software Operation measurement result list in the List. If the users want to see more detail absorbance intensity information, they can see them in Show Detail in List. The absorbance intensity data can be stored in Excel file by clicking To Excel. When click My data button to open the file, the measurement result and absorbance curve will be restored in the software. 6) When the measurement is complete, a new laboratory wipe should be used to wipe the pedestals. In this way, the users can do next measurement. If measure the same lot samples, the users do not need to re-blank. It is advisable to do the blanking every 15min at least. 7) The software automatically adds the measurement results into the measurement results list in List. It is best to click 'save' button to save them in the user-specified region before closing the software, since the data will disappear when closing the software. 5. Debug Mode In the Task bar, click "Options" or choose "DebugEnable" item from "Debug", then the software enter into Debug mode. In that time, the setting keys for user references will be added in the software interface. It includes: 1) Debug menu Dropdown menu of Debug incudes: DebugEnable: select if enter into Debug mode or not. LampEnable: select if open Asynchronous switch lamp. R_EliminateBlackI: select if eliminate black current ContinueBlank: select if automatically continue blanking without clicking. ContinueMeasure: select if automatically cycle measure without clicking. SaveBlankData: save the blank data for this measurement 2) Debug tool bar 21

27 Chapter 5 Software Operation The user can set references to satisfy specific measurement requires. If there is any question, or you have any other requirement for the reference key, you can contact the manufacturer. Inside the Debug tool bar includes: Integration time: the integration time for CCD sensor test. The shortest time is not less than 25min. isamplenum: the average value for CCD sensor data Average Num: the average value Boxcar width: do the smooth processing for absorbance curve of specific wavelength Boxcar Num: the times to do smooth processing for absorbance curve. Note: Above set data will be active after clicking "Input" 3) Baseline correction checkbox Inside the baseline correction checkbox includes: Baseline correction: select if do baseline correction Select wave: select the correction wave 22