CN207488183U - Near-infrared spectrometer and transmission platform - Google Patents

Abstract

The utility model relates to a transmission platform of a near-infrared spectrum detector, which comprises a base plate, on which two first vertical plates and second vertical plates parallel to each other are arranged, and each of the first vertical plate and the second vertical plate is provided with a There are corresponding transmission windows, the transmission windows are covered with optical windows, and the optical windows are located on the opposite sides of the two vertical plates, and a stage is arranged between the first vertical plate and the second vertical plate for carrying the container. There is a soft bag for the object to be tested, and the stage is located below the lower edge of the optical window, the soft bag containing the object to be tested is located between the two optical windows, and at least one vertical plate can move horizontally along the surface of the substrate , so that the soft bag containing the object to be measured is clamped between two optical windows and the optical path between the two optical windows can be adjusted. The utility model also relates to a near-infrared spectrometer comprising the above-mentioned transmission platform.

Description

Near-infrared spectroscopy detector and transmission platform

technical field

The utility model relates to the field of biological detection instruments, in particular to a near-infrared spectrum detector and a transmission platform.

Background technique

In order to prevent virus infection from blood transfusion, the methylene blue light method is widely used to inactivate plasma virus in clinical plasma. This is the only virus inactivation technology approved for clinical use in a single blood component in my country. The inactivation process is carried out in the plasma inactivation bag, and the release of the inactivator methylene blue in the plasma inactivation bag enters with the plasma through gradual dissolution when the plasma flows through the methylene blue storage under sterile conditions Inactivation bag, this process is the release process of methylene blue, and its release amount is a key parameter to determine the degree of virus inactivation. A large number of data show that the virus inactivator methylene blue must be released within a very narrow range, which is 0.9-1.3 μmol/L in my country. In order to detect the release amount as conveniently, quickly and sensitively as possible, a large number of sampling-based detection methods have emerged, including solid phase extraction-spectrophotometry, enhanced fluorescence spectroscopy, HPLC, chemiluminescence, and resonance Rayleigh scattering spectroscopy. Law. Sampling detection requires the use of extraction devices to extract samples from the plasma inactivation bag for testing. Since the extraction operation will cause damage to the plasma inactivation bag, this destructive detection method can easily lead to secondary contamination of plasma during sampling, including bacteria, chemical Pollutants, as well as particulate matter, cause the plasma in the plasma inactivation bag to no longer be used in the human body, resulting in a waste of plasma. It is also based on this that the plasma that has been tested for release can no longer be used clinically. Therefore, it is impossible to carry out such inactivation quality control based on destructive detection for each bag of plasma used in clinical practice.

At present, near-infrared spectroscopy is usually used for non-destructive and rapid detection of samples, which is suitable for quality control in the production process. Among them, diffuse reflectance near-infrared spectroscopy should be the most widely used for non-destructive testing of samples, but this method has certain limitations: firstly, this method is generally applicable to solid, powder, and paste, but it is difficult to apply to liquid samples. After the light is irradiated into the liquid, only extremely weak diffuse reflection light is produced, and most or even all of it is transmitted light, so it is difficult to quantitatively detect a certain substance in the liquid based on its diffuse reflection near-infrared spectrum.

Due to the structural limitations of existing near-infrared infrared spectrometers, the existing near-infrared spectroscopy detection method for rapid quantitative detection of substances in liquids usually requires sampling from the container to be detected, which mainly includes two methods. The first one is to place the obtained liquid sample in the cuvette, and then put it into the cuvette rack of the instrument for detection; the second one is to insert the liquid optical fiber probe with a slit into the liquid in the container to be detected The detection liquid flows directly into the slit of the optical fiber probe as a cuvette to realize the sampling operation, and the optical fiber transmits the signal to the spectrometer to realize fast detection. No matter which one of the two methods is used, it is a destructive detection method, and the defects of this method have been described in detail above. However, using the existing infrared spectrometer to directly detect the plasma bag will encounter great difficulties. First, the plasma inactivation bag contains many air bubbles. If these air bubbles are in the detection area, the measurement results will have a large error; secondly , when the plasma inactivation bag is directly detected, because the bag body is soft and its shape is irregular, the optical path of the spectral detection is difficult to fix, and it is difficult to calculate the content of the analyte using a quantitative formula. Of course, the plasma in the inactivation bag contains a large number of biological macromolecules, and the carbon-hydrogen bonds in it will generate a large number of near-infrared spectrum signals, which will also interfere with the detection results and bring certain difficulties to the detection method.

Utility model content

In order to solve the above-mentioned technical problems, the purpose of this utility model is to provide a near-infrared spectrum detector and a transmission platform. Non-destructive, rapid and accurate analysis of methylene blue release from inactivation bags.

The utility model provides a transmission platform of a near-infrared spectrum detector. The transmission platform includes a base plate, on which two first vertical plates and second vertical plates parallel to each other are vertically arranged, and the first vertical plate and the second vertical plate There are corresponding transmission windows on each of the vertical plates, and the transmission windows are covered with transparent optical windows, and the optical windows are located on the opposite sides of the two vertical plates. The object table is used to carry the soft bag containing the object to be tested, and the object table is located below the lower edge of the optical window, the soft bag containing the object to be measured is located between the two optical windows, and at least one vertical plate It can move horizontally along the surface of the base plate, so that the soft bag containing the object to be tested is clamped between two optical windows and the optical path between the two optical windows can be adjusted.

Further, the stage is located at least 2cm below the lower edge of the optical window.

Further, the first vertical plate is fixedly connected with the base plate, the loading table is fixedly connected with the first vertical plate, and the second vertical plate is provided with a strip-shaped hole facing the loading table, and the strip-shaped hole is consistent with the shape of the loading table. Fitting, the stage slides into the bar hole.

Furthermore, a guide rail is fixedly connected to the base plate, and the second riser can slide horizontally along the guide rail and can be locked at any position of the guide rail, thereby fixing the distance between the two optical windows (that is, the optical path).

Further, there are two guide rails, and each guide rail is arranged perpendicular to the second vertical board.

Further, a positioning platform is fixedly connected to the side of the second vertical plate away from the first vertical plate, the second vertical plate is slidably connected to the guide rail through the positioning platform, and the positioning platform is screwed with a screw rod, and the axial direction of the screw rod is the same as that of the second vertical plate. Vertically, a fixed baffle is fixedly connected to the guide rail, the fixed baffle is provided with a through hole, and the screw rod is passed through the through hole.

Further, the material of the optical window is quartz or optical glass.

Further, the transmission window is rectangular.

Further, the area of the optical window is larger than that of the transmission window.

Further, the stage is arranged parallel to the substrate.

The utility model also provides a near-infrared spectrometer, including a light source, a monochromator, a detector, a computer processing information system, and the above-mentioned transmission platform.

By means of the above scheme, the utility model has at least the following advantages:

The structural setting of the transmission platform of the near-infrared spectrum detector of the utility model can realize the fixed optical path during the non-destructive detection of the plasma inactivation bag, and can effectively solve the problem that the air bubbles in the plasma inactivation bag exist in the detection optical path, so that the quantitative The results are accurate and reliable. Due to the extrusion of the optical windows, the plasma inactivation bag between the two optical windows is squeezed into a regular plane. The air bubbles in the plasma inactivation bag between them are naturally driven to the plasma inactivation bag outside the extrusion part of the optical window, so that the air bubbles will not appear in the detection area.

When the near-infrared spectrum detector containing the transmission platform of the utility model is used, it can realize the non-destructive, fast and accurate measurement of the amount of methylene blue released in the plasma inactivation bag outside the plasma inactivation bag in the non-sampling mode analyze.

The above description is only an overview of the technical solution of the utility model. In order to understand the technical means of the utility model more clearly and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiment of the utility model with accompanying drawings. back.

Description of drawings

Fig. 1 is the three-dimensional structure schematic diagram of the transmission platform of the present invention;

Fig. 2 is a schematic diagram of the front side structure of the transmission platform of the present invention;

Fig. 3 is a schematic diagram of the structure of the left side of the transmission platform of the present invention;

Fig. 4 is a schematic diagram of an enlarged structure of circle A in Fig. 3;

Fig. 5 is a top view structural schematic diagram of the transmission platform of the present invention;

Fig. 6 is the near-infrared spectrogram after baseline drift is eliminated in embodiment 2;

Figure 7 is the residual of the PLS model based on the leave-one-out interactive test;

Fig. 8 is the multivariate analysis model figure of methylene blue release amount;

Explanation of reference signs:

1-second vertical plate; 2-quartz plate; 3-plasma inactivation bag; 4-transmission window; 5-substrate; 6-first vertical plate; 7-stage; 8-positioning platform; 9-screw; 10-guide rail; 11-fixed baffle.

Detailed ways

Below in conjunction with accompanying drawing and embodiment, the specific embodiment of the utility model is described in further detail. The following examples are used to illustrate the utility model, but not to limit the scope of the utility model.

Example 1

Referring to Figures 1-5, a transmission platform of a near-infrared spectrum detector of the present invention includes a base plate 5 on which two first vertical plates 6 and a second vertical plate 1 parallel to each other are movably connected. A riser 6 and a second riser 1 are arranged perpendicular to the base plate 5 . Each of the two vertical plates is provided with a corresponding rectangular transmission window 4, and the upper cover of the transmission window 4 is provided with a square transparent quartz plate 2. The quartz plate 2 is located on the opposite side of the two vertical plates, and the area of the quartz plate 2 is larger than that of the transmission window. The area of window 4. The first vertical plate 6 is fixedly connected with the base plate 5, and the first vertical plate 6 is also fixedly connected with a stage 7, the stage 7 is located at least 2 cm below the lower edge of the quartz plate 2, and the stage 7 is used for placing Soft bag for measuring objects. The upper surface of the stage 7 is parallel to the base plate 5, and the second vertical plate 1 is provided with a strip hole facing the stage 7, and the strip hole is adapted to the shape of the stage 7, so that the loading Table 7 is slidably inserted into the bar-shaped hole. Two guide rails 10 are fixedly connected to the base plate 5 , the guide rails 10 are arranged perpendicular to the second riser 1 , and the second riser 1 can slide horizontally along the guide rails 10 . The second riser 1 is fixedly connected with a positioning platform 8 on one side away from the first riser 6, and the positioning platform 8 is threadedly connected with a screw rod 9, and the axial direction of the screw rod 9 is perpendicular to the second riser 1, and the positioning platform 8 and the guide rail 10 Slidingly connected, the guide rail 10 is fixedly connected with a fixed baffle 11, the fixed baffle 11 is provided with a through hole, and the screw rod 9 is penetrated in the through hole.

When performing sample detection, such as the sample detection in the plasma inactivation bag 3, first adjust the screw to drive the second vertical plate 1 to move along the guide rail 10, so that it slides to the required distance, and the sliding direction is perpendicular to the plane where the vertical plate is located , and then adjust the screw 9 to fix the second vertical plate 1 at this position, that is, the optical path of the transmission detection is fixed. And during the sliding process of the second vertical plate 1, the stage 7 is slidably inserted into the strip-shaped hole provided thereon to facilitate the adjustment of the optical path and ensure that the quartz plate 2 can sterilize the plasma under different optical paths. Live bag 3 is clamped. Put the plasma inactivation bag 3 on the stage 7, and squeeze it slightly above the bag, so that the plasma inactivation bag is in close contact with the two optical windows, so that the plasma inactivation bag 3 is clamped between the two optical windows. Between 2 pieces of quartz. When the quartz plate 2 squeezes the plasma inactivation bag 3, the plasma inactivation bag 3 between the two quartz plates 2 forms a regular shape, thereby fixing the exact optical path (marking line "5" in Fig. 4 "The distance marked is the detection optical path), and the air bubbles in the plasma inactivation bag 3 migrate to the part of the plasma bag above the upper edge of the quartz sheet 2, so that there are no air bubbles between the quartz sheets 2, preventing it from affecting the detection results .

Example 2

In the following examples, the materials used include: non-inactivated human plasma provided by the Blood Bank of Suzhou Red Cross Center, using the filter in the disposable virus inactivation accessory device to filter out white blood cells to obtain plasma without methylene blue ( i.e. blank plasma). Disposable blood collection and transfusion set containing plasma inactivation bag (transfer bag, specification: 35ml, production batch number: 170428, Sichuan Nangeer Biotechnology Co., Ltd.). Methylene blue hydrochloride (analytically pure, >97% based on dry product content, Sigma-Aldrich Company), the experimental water is triple distilled water. Near-infrared spectrometer (model: NEXUS, American Thermo Company), its transmission platform is modified according to embodiment 1.

Using the near-infrared spectrometer that contains the transmission platform of the present utility model, first set up the multivariate analysis model of methylene blue release, specifically as follows:

(1) First prepare the methylene blue stock solution: weigh 1.3953 g of methylene blue (analytically pure, with a drying weight loss rate of 10.6%), place it in a 500 mL volumetric flask, and dilute to volume with PBS solution. Accurately measure 1.00ml of methylene blue solution, and then dilute to 1000ml with PBS solution to obtain 7.8μmol/L methylene blue stock solution. Protect from light and keep sealed.

(2) Reconfigure the sample to be tested: firstly, use the methylene blue stock solution and PBS buffer solution (pH=7.4) to prepare primary and secondary dilutions by stepwise dilution method. After that, a total of 52 methylene blue plasma solutions with a concentration of 0.312-2.959 μmol/L were precisely prepared from the stock solution, dilution solution and plasma. Finally, inject the methylene blue plasma solution into each blood collection and transfusion transfer bag with a disposable sterilized syringe to obtain the sample to be tested.

(3) Near-infrared spectroscopy detection of samples

Wipe the outer surface of the plasma inactivation bag to be tested and the quartz plate on the transmission platform with alcohol, and adjust the distance between the first vertical plate 6 and the second vertical plate 1 according to the method in Example 1, so that the distance between the two optical windows is The optical distance between them is fixed at 3.5mm. Afterwards, the plasma inactivation bag was placed on the transmission platform according to the method in Example 1, and the position of the plasma inactivation bag was aligned with the left and right sides as far as possible each time, and the spectrum of the sample was measured. The instrument parameters are: wavenumber 4000-10000cm ^-1 , optical path 3.5mm, resolution 8cm ^-1 , scanning times 32, scanning speed 0.3165, aperture 22, gain 1. Air-filled, unused plasma inactivated bags were used as spectral background before testing samples on the day.

(4) The near-infrared transmission spectrum of the sample in the plasma bag obtained by detection is used as the standard spectrum in the range of 3999.7-4265.8 cm ^-1 to perform baseline drift calibration. Select 5827.9-6464.3cm ^-1 and 7467.1-9009.9cm ^-1 as the rich information interval, take the mean-centered spectrum as the model variable, select the number of principal components as 11, and randomly select 11 samples according to the distribution of concentration as the prediction set. Taking the remaining 41 samples as the calibration set, the multivariate analysis model of methylene blue release was established by PLS method.

For the low wavenumber region of the near-infrared spectrum, there will usually be a spectral baseline. It should be a horizontal line, and the spectra of samples of different concentrations in this interval should completely overlap, have no statistical difference, and have an absorbance mean of 0. In Fig. 6, the spectral baseline of each sample is in the interval of 3999.7-4265.8 cm ^-1 . However, in the original spectrum, the baselines of samples with different concentrations drifted by about 7, and this concentration-independent drift will affect the accuracy of methylene blue release. The average absorbance value of the detected spectrum of each sample in the range of 3999.7-4265.8 cm ^-1 is used as the drift amount, and the drift is eliminated to obtain the drift-eliminated spectra of all samples.

From the spectrum after baseline drift elimination (Figure 6), it can be found that the plasma sample of methylene blue has two obvious absorption peaks in the range of 5350-6600cm ^-1 and 7200-10001cm ^-1 in the entire spectral range, which are the absorption peak spectral regions . For the former, it consists of two high and low shoulder peaks. In the low wave number region of 5350-5828cm ^-1 , the spectrum not only has low absorption, but is basically a horizontal line without peaks, and there are wavy burrs. This type of low signal-to-noise ratio The spectral signal will affect the precision of the quantitative analysis model. The spectrum in the high wavenumber range of 6464-6600cm ^-1 is the end spectrum of the absorption peak, with low absorption and weak signal, which also shows a low signal-to-noise ratio. Therefore, the spectrum at 5827.9-6464.3 cm ^-1 in the absorption peak spectral region is selected as the signal-rich spectrum. Similarly, in the 7200-10001cm ^-1 absorption peak spectral region, select the 7467.1-9009.9cm ^-1 spectrum as the rich signal spectrum.

Using the spectra of 5827.9-6464.3cm ^-1 and 7467.1-9009.9cm ^-1 as the rich information spectrum, using the mean-centered spectrum as the model variable, and using the calibration set samples according to the leave-one-out interactive test, calculate the scores of different principal components When the PLS model predicts the residuals of methylene blue concentration (Fig. 7). We can find that as the number of principal components increases, the predicted concentration residuals tend to decrease gradually, indicating that the accuracy of the model gradually increases. When the number of principal components is greater than or equal to 11, the degree of reduction in the prediction residual decreases significantly, indicating that the continued increase in the number of principal components has a low contribution to the accuracy of the model. Of course, if the number of principal components is too large, it will also lead to overfitting of the model. Therefore, 11 is selected as the number of principal components.

Taking the information-rich spectra of the selected two intervals as the identification variable of the release of methylene blue, based on the previously determined principal component numbers, centering the spectral mean, and using the PLS method to establish a multivariate analysis model for the release of methylene blue, in which methylene blue For the high and low distribution of blue concentration, 11 samples are randomly selected as the prediction set, and the remaining 41 samples are the prediction set. See Figure 8 for the results. Represents the calibration set samples, ● represents the prediction set samples. It can be found that in the multivariate analysis model, the calibration concentration and the real concentration of the calibration sample can correspond to each other well, and they are all distributed near the real concentration (diagonal line) in the entire detection concentration interval, and the correlation coefficient Rcorr=98.16 %. Using the established model to predict the release of methylene blue in the independent prediction set samples (i.e. sample detection), it is not difficult to find that the predicted concentrations of these samples still show a good correspondence, and they can also be distributed in Near the real concentration, the correlation coefficient Rpred is as high as 98.93%. This shows that the model established by the utility model has good accuracy.

In order to illustrate the accuracy of the model, the samples of high, middle and low concentration groups were designed and the recovery rate test was carried out. The results are shown in Table 1. It can be seen from Table 1 that no matter the concentration is high or low, each concentration group has a good recovery rate, the recovery rate is 103.6%-110.8%, and there is no correlation between the concentration level and randomness, which shows that the established non-destructive testing method is accurate and reliable. Of course, the lowest recovery rate is as low as 96%, and the highest is as high as 119%, which is an acceptable accuracy for a non-destructive and rapid detection method, especially for the detection of biological samples.

Table 1 Accuracy of non-invasive detection of methylene blue release in plasma by near-infrared spectroscopy

Above result shows, adopt the transmission platform of near-infrared spectrometer of the present utility model, utilize the good penetrability of near-infrared light, adopt near-infrared transmission spectrum to set up the plasma virus inactivator methylene blue discharge amount in the blood collection and transfusion transfer bag Nondestructive testing models and methods. The near-infrared transmission spectrum of the sample in the plasma bag obtained by non-destructive testing outside the plasma bag was used as the identification variable, and the baseline drift calibration was performed based on the spectrum in the interval 3999.7-4265.8cm ^-1 , and 5827.9-6464.3cm ^-1 and 7467.1- The spectrum in the 9009.9cm ^-1 interval is an information-rich spectrum, the mean-centered spectrum is used as the model variable, the number of principal components is selected as 11, 11 samples are randomly selected as the prediction set, and the remaining 41 samples are used as the correction set, and the PLS Methods A multivariate analysis model for methylene blue release was established. The formula for this model is as follows:

c=k ₁ A ₁ +k ₂ A ₂ +···+k _n A _n , where c represents the concentration of methylene blue in the sample, A ₁ , A ₂ ···A _n are the detected The wavelengths corresponding to the selected information-rich spectral intervals in the spectrum are the absorbance at λ ₁ , λ ₂ ···λ _n , and k ₁ , k ₂ ···k _n are the corresponding PLS regression coefficients.

The correlation coefficients of the model-calculated concentration to each sample concentration were 98.16% (calibration set) and 98.93% (prediction set), and the average recoveries of samples in the high, middle and low concentration groups were 103.6%-110.8%. This shows that the near-infrared transmission spectroscopy method established by the utility model can realize the non-destructive, fast and accurate analysis of the release amount of methylene blue in the collection and transportation transfer bag outside the plasma bag. It indicates that under the condition of no secondary pollution, the monitoring of the quality of plasma virus inactivation in the collection, transportation and transfer bag can be realized.

Example 3

Use the near-infrared spectrometer that contains the transmission platform of the present utility model, adopt the multivariate analysis model of the methylene blue release amount of above-mentioned establishment to carry out the detection of methylene blue release amount in blood plasma, specifically as follows:

Wipe the outer surface of the plasma inactivation bag to be tested and the quartz plate on the transmission platform with alcohol, adjust the distance between the first vertical plate 6 and the second vertical plate 1 according to the method in Example 1, so that the distance between the two optical windows The optical distance between them is fixed at 3.5mm. Then put the plasma inactivation bag with unknown methylene blue content (that is, the plasma inactivation bag that released methylene blue) on the stage, and align the left and right sides of the plasma inactivation bag each time as much as possible, and measure the sample spectrum. The instrument parameters are: wavenumber 4000-10000cm ^-1 , optical path 3.5mm, resolution 8cm ^-1 , scanning times 32, scanning speed 0.3165, aperture 22, gain 1. Air-filled, unused plasma inactivated bags were used as spectral background before testing samples on the day. Then the measured near-infrared transmission spectrum is calibrated with the spectrum in the range of 3999.7-4265.8 cm ^-1 as the standard for baseline drift. Select 5827.9-6464.3cm ^-1 and 7467.1-9009.9cm ^-1 as the rich information interval, and substitute the absorbance at each wavelength in this interval into the formula of the model established in Example 2 to calculate the methylene blue in the plasma inactivation bag content.

The above is only a preferred embodiment of the utility model, and is not intended to limit the utility model. It should be pointed out that for those of ordinary skill in the art, they can also do Several improvements and modifications are made, and these improvements and modifications should also be regarded as the protection scope of the present utility model.

Claims (10)

1. a kind of transmission platform of near-infrared spectrometer, it is characterised in that：The transmission platform includes a substrate, the base Two pieces of starting staves being mutually parallel and the second riser are equipped on plate vertically, is respectively offered on the starting stave and the second riser Corresponding transmissive window, the transmissive window is covered with transparent optics window, and to be located at two pieces of risers opposite for the optics window Side on, an objective table is equipped between the starting stave and the second riser, the soft bag of determinand is filled for carrying, it is described Objective table is located at the lower section of optical window piece lower edge, and the soft bag for filling determinand is located between two optics windows, and at least one A riser can be moved horizontally along the surface of the substrate, and the soft bag for filling determinand is made to be clamped between two optics windows And the light path between two optics windows can adjust.

2. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：The starting stave with The substrate is fixedly connected, and the objective table is fixedly connected with the starting stave, and face institute is offered on second riser The bar hole of objective table is stated, the bar hole is adapted with the shape of the objective table, and the objective table can be slidably inserted into described In bar hole.

3. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：It is fixed on the substrate Several guide rails are connected with, second riser can slide along the guide rail level and can be locked in any position of guide rail.

4. the transmission platform of near-infrared spectrometer according to claim 3, it is characterised in that：The number of the guide rail It it is two, each guide rail is set perpendicular to second riser.

5. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：Second riser is remote From a positioning table is fixedly connected in the one side of starting stave, second riser is slided by the positioning table and guide rail to be connected It connects, the positioning table is threaded with a screw rod, and the axial direction of the screw rod and second riser are perpendicular, solid on the guide rail Surely a fixed baffle is connected with, the fixed baffle opens up a through-hole, and the screw rod is arranged in the through-hole.

6. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：The optics window Material is quartz or optical glass.

7. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：The transmissive window is in square Shape.

8. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：The optics window Area is more than the area of transmissive window.

9. the transmission platform of near-infrared spectrometer according to claim 1, it is characterised in that：The objective table is parallel It is set in the substrate.

10. a kind of near infrared spectrometer exists including light source, monochromator, detector and computer treatmenting information system, feature In：Further include the transmission platform described in any one of claim 1-9.