UV/Vis capable Multiskan Sky Microplate Spectrophotometer with touch screen

Exceptional user experience

The Multiskan SkyHigh Microplate Spectrophotometer is a UV/Vis microplate spectrophotometer designed to be convenient and easy to use for virtually any photometric research application, especially DNA, RNA, and protein analysis, as well as turbidity measurements. It is ideal for multi-user environments where a variety of endpoint, kinetic, and spectral assays are performed. Multiskan SkyHigh instruments are available in three different configurations. Touch screen models offer the flexibility to use the stand-alone instrument or in conjunction with Thermo Scientific SkanIt PC software. The Multiskan SkyHigh model operated solely via SkanIt software is ideal for users who rely on a PC for all operations. Cuvette reading capability is also offered in some models.

Key features

Fast, easy nucleic acid and protein sample measurements in low volumes

Our µDrop and µDrop Duo Plate is designed for rapid measurement of low sample volumes. You can analyze up to 16 samples simultaneously. Users can quickly and easily wipe off the samples for serial measurements. SkanIt software sessions are available for nucleic acid analysis. The μDrop Plate also contains a dedicated holder for cuvette measurements.

Simple-to-use software enables experiment setup within minutes

SkanIt software can be used to control the Multiskan SkyHigh spectrophotometer and supports optimal use of the instrument’s features with a visual workflow, easy data analysis, and exporting capabilities. SkanIt software is a multi-instrument microplate reader software that provides excellent usability and flexibility even for the most challenging assays.

Access and manage your data remotely

The Multiskan SkyHigh instrument touch screen models can connect to Thermo Fisher Connect and Microsoft OneDrive cloud-based tools to help ensure secure digital data management, permit remote access to your photometric data, and allow sharing between colleagues—all from the comfort of your computer.

Thermo Fisher Connect cloud-based capabilities enable you to:

  • Monitor the status of your instrument from your PC or Mac® computer.
  • Monitor the status of your instrument from your mobile device with the Instrument Connect app.
  • Monitor the progress of a kinetic run (e.g., bacterial growth) from your cloud account or from your mobile device with the Instrument Connect app.
  • Upload measured data automatically or manually from your instrument to your cloud account.
  • Securely store, access, share, and manage your data remotely.
  • Receive automatic notification of new instrument software updates.
  • Upgrade instrument software at your convenience.
     

Technical specifications

Wavelength selection Monochromator
Light source Xenon flash lamp
Wavelength range 200–1,000 nm with 1 nm steps
Read-out range Up to 4 absorbance units
Bandwidth <2.5 nm
Linearity at 450 nm* 0–2.5 Abs, 2%
Accuracy at 450 nm* 1.0% + 0.003 Abs (0–2.0 Abs)
2.0% (2.0–2.5 Abs)
Precision at 450 nm* SD <0.003 Abs or CV <1.0%
Plates 6-48, 96, and 384 well plates†, Thermo Scientific µDrop Plates
Cuvette Dimensions: 12.5 (W) x 12.5 (D) x 40-58 (H) mm
Beam center height: 8.5 mm
Beam window: ≥2 mm
Measurement speed (from A1 back to A1) 6 s with 96-well plate
10 s with 384-well plate
Plate shaking Linear
Spectral scanning speed 10 s from 200–1,000 nm with 1 nm steps
Incubation range From ambient + 2°C to 45°C
User interfaces Stand-alone use: 7 in. touch screen display
PC control: SkanIt software
Connections 1 USB B port for PC
1 Ethernet port
3 USB A ports for devices (USB memory device and Wi-Fi dongle)
Main input 100–240 V (50/60 Hz)
Max power consumption <110 W
Power save consumption** 7 W
Dimensions (H x W x D) 265 x 295 x 445 mm [10.4 x 11.6 x 17.5 in]
Weight 11.3 kg [24.9 lbs]
* Specifications are valid for 96-well flat bottom microplates and quartz semimicro cuvettes with a 4 mm beam window.
** Models with touch screen. †Maximum plate height with lid is 19.5 mm.

Low-volume quantification of nucleic acids and proteins with Thermo Scientific µDrop and µDrop Duo Plates

Introduction

Photometry in the UV range is a common way to quantify nucleic acids and proteins in a sample. Both nucleic acids and proteins absorb UV light. In the case of nucleic acids, the nitrogenous bases present in all nucleotides have a clearly distinguishable absorption maximum at 260 nm. On the other hand, in the case of proteins, the light absorptive properties at 280 nm are limited to few amino acids, specifically the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr), and to a lesser extent cysteine groups forming disulfide bonds (Cys-Cys). Therefore, the absorption of proteins and peptides at 280 nm is proportional to the content of these amino acids.

These UV-absorptive properties make it possible to use direct photometry to quantify the concentrations of nucleic acids and proteins. Such calculations are performed using Beer-Lambert’s Law, which describes that the absorbance of a certain nucleic acid or a protein depends on the molecule’s concentration, the molecule’s absorptivity coefficient (given by the intrinsic absorptivity properties of such molecule) and the pathlength of the incident light. The use of this equation specifically for calculation of nucleic acids and proteins is shown on Table 1.

Table 1. Calculation of nucleic acids and protein concentrations using direct absorbance measurements, according to Beer-Lambert’s Law.

  Nucleic acids Proteins
Example case Double stranded DNA (dsDNA) Bovine serum albumin (BSA)
Equation for the calculations

Where:
C = concentration of dsDNA (µg/mL)
A260 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
εdsDNA = extinction coefficient of dsDNA = 0.02 (µg/mL)-1cm-1
Note: Ratio of is 50 (µg/mL) cm

Where:
C = concentration of BSA (mg/mL)
A280 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
ε1% = mass extinction coefficient of BSA = 6.7 (mg/mL)-1cm-1 for 1% (10 mg/mL) BSA solution

Equation for molar extinction coefficient for ε proteins   Molar extinction coefficient ε for any protein can be easily calculated using following equation:
ε=(nW * 5500) + (nY * 1490) + (nC * 125)
Where:
W: tryptophan, Y: tyrosine, C: cysteine
n: number of each residue present in the protein
(5500, 1490, and 125: molar absorptivity at 280 nm of W, Y, and C, respectively)

The calculations of nucleic acids and proteins are performed based upon absorbance measurements at 260 nm and 280 nm, respectively, but an additional measurement at 320 nm is typically carried out with the purpose of background subtraction. Measuring at 320 nm allows excluding impurities in the sample, for example arising from the presence of magnetic beads in the sample.
 

Low-volume quantification with Thermo Scientific µDrop Plate and µDrop Duo Plates

The amount of sample available for nucleic acid and protein analysis is often low. To address that challenge, Thermo Scientific offers the µDrop and µDrop Duo Plates, which enable direct photometric measurements at a microliter scale (Figure 1). The Drop and µDrop Duo Plates consist of two separate measurement locations: the left side is intended for measurements of low-sample volumes and the right side is for measuring cuvettes. The cuvette slot is used to perform photometric measurements (including kinetic studies) with standard cuvettes that are covered with stopper and placed in a horizontal position.

The low-volume measurement area consists of two or four quartz slides: one or two pairs of the top clear quartz slide and the bottom quartz slide, which is partially Teflon-coated. The bottom slide contains a matrix array position where samples are pipetted. In the case of the µDrop Plate, there are 16 samples positions arranged in a 2 x 8 matrix, while in the case of the µDrop Duo Plate, there are 32 sample positions arranged in 2 separated arrays also in a 2 x 8 matrix (Figure 1). The pathlength of the µDrop Plate is fixed, and the nominal value is 0.05 cm. However, the pathlength can vary between 0.048 and 0.052 cm, and verified pathlength for each μDrop Plate is indicated on the quality control measurement report delivered with the μDrop Plate. Same applies to the µDrop Duo Plate (with 32 samples), but in this case, there are two separate pathlength values, one for each 2 x 8 array. These exact pathlength values should always be used for the concentration calculations, instead of the nominal value of 0.05 cm, in order to obtain more accurate results.

top view of the plates showing the quartz slides and the positions for pipetted samples

Figure 1. μDrop Plate and the μDrop Duo Plate.

Compared to a normal cuvette having a pathlength of 1 cm, the pathlength of the μDrop Plate or μDrop Duo Plate is 20 times shorter (0.05 cm). Because of this, there is roughly a 20 times difference between the concentrations that can be measured with a typical cuvette versus the μDrop and μDrop Duo Plate. This means that the detection limit of any given analyte is about 20 times higher in the μDrop and μDrop Duo Plate than with cuvettes, when measured in the same instrument. On the other hand, a shorter pathlength allows for lower sample volumes to be measured. In the case of the μDrop and μDrop Duo Plate, a sample volume down to 2 µL can be used. It is possible to measure nucleic acid and protein concentrations from a few to thousands of micrograms per microliter using these devices together with a photometer that has sufficiently high precision and a wide linear range, such as the Thermo Scientific Multiskan SkyHigh Microplate Spectrophotometer. Finally, any photometric measurement device always has a certain background absorption. Therefore, blank subtraction is necessary when photometric quantification of the sample concentrations is performed.


Materials and methods

To calculate the detection limits for nucleic acid quantification (dsDNA), 11 dilutions were made of a DNA stock solution of Herring sperm DNA (Promega, D1816) using TE-buffer, (Invitrogen 10 mM Tris-HCl, 0.1 mM EDTA), to cover concentration range between 50 and 3500 µg/mL. To calculate the detection limits for protein quantification (BSA), 9 dilutions were made of a BSA stock solution (Sigma, A7030) to distilled water to cover concentration range between 0.5 and 100 mg/ml. The blanks as well as dsDNA or BSA samples were measured by pipetting 4 µL into the measurement areas of the μDrop Plate and the µDrop Duo Plate. In case of both μDrop and µDrop duo plates, 2 blanks were used. All measurements were performed using Multiskan SkyHigh Microplate Spectrophotometer with touchscreen and cuvette.

As indicated earlier, to ensure that proper concentration calculations, the exact pathlength values of the μDrop Plate or µDrop Duo Plate need to be provided. These values were defined in Multiskan SkyHigh settings by selecting “Settings” from the Home screen and then selecting “μDrop Plates” as indicated in Figure 2. The final concentrations reported by the instrument will be calculated with these pathlength values, and the user will not need to perform any correction steps, afterwards. Ready-made assay protocols in the Multiskan SkyHigh user interface for dsDNA or BSA quantification were used to perform the assays.

User interface screenshot showing how users specify pathlengths into the software

Figure 2. Setting-up the pathlength values of the μDrop Plate and μDrop Duo Plate in the User Interface of the Multiskan SkyHigh Microplate Spectrophotometer.

When the Multiskan SkyHigh model without touch screen is used, the dsDNA concentration calculations need to be done using the Thermo Scientific SkanIt software, which controls all the Thermo Scientific microplate readers. For that purpose, in “Plate Layout”, the appropriate plate format should be selected from the dropdown menu (μDrop Plate or μDrop Duo Plate). In the case of the μDrop Duo Plate, it is possible to add two values as the pathlengths on the two separate measurement areas may differ. See an example of the performed calculations in Figure 3.

Screenshot of software showing the results of the dsDNA concentration calculations

Figure 3. Setting-up the pathlength values of the μDrop Duo Plate in the SkanIt software for calculations of the dsDNA concentrations using Multiskan SkyHigh Microplate Spectrophotometer. If the calculation is performed without 320 nm subtraction, the factor 50 is simply added to the pathlength correction step of the 260 nm measurement data.

Multiskan SkyHigh models with UI, the ready-made sessions have all the necessary calculations. The results automatically contain the concentration, purity ratios and the sample spectrum, Figure 4 below.

Calculations results screen showing the dsDNA measurements and corresponding concentration and purity values

Figure 4. An example data set of a µDrop duo plate dsDNA measurement.


Results and discussion

Detection range of the µDrop Plate and µDrop Duo Plates

The detection range is given by the lowest and highest possible concentrations that can be reliably measured with a given instrument. Therefore, these two extremes of the detection range are instrument dependent and they can be theoretically calculated from the instrument’s performance specifications. In this note, however, we focus on the detection ranges of nucleic acids and proteins, as experimentally measured using IUPAC recommendations. Often the limiting factor is the lowest end of the detection range, since unknown samples that have too low concentrations of nucleic acids or proteins (falling under the lowest part of the detection range) cannot be simply accurately estimated using photometric detection with the µDrop Plate or µDrop Duo Plates. Often, because most microplate spectrophotometers in the market have very similar detection ranges, the only solution in such cases is to change the detection technology and use instead fluorescence-based detection, which is known to be considerably more sensitive. By contrast, for samples with too high concentration values (falling outside of the detection range), a simple solution is to dilute the samples, so that they can fall within the detection range, and thus can be accurately measured.

Limits of Detection (LOD) and Limits of Quantification (LOQ) for measuring nucleic acids and proteins

The lowest part of the detection range curve indicates the assay sensitivity, which can be assessed, according to the IUPAC, with two different parameters: Limit of Detection (LOD) and Limit of Quantification (LOQ). The LOD is defined as the lowest quantity or concentration of analyte that can be separated from the background or blank values. The LOD value means that the presence of the analyte can be detected with statistical significance, but the analyte cannot be quantified as an exact value. On the other hand, the LOQ is defined as the lowest quantity or concentration of the analyte at which quantification is possible with statistical relevance. In practice, LOD is the limiting value in qualitative assays where the question “is there any analyte in my sample or not?” needs to be answered. In such cases, LOQ is the limiting value that the user can measure as the concentration of the analyte in quantitative assays. Both LOD and LOQ calculations assume that both blank samples and replicates of the analyte are normally distributed. Signals at the LOD are defined as the blank mean + 3 * standard deviation of the blank, while signals at the LOQ are defined as blank mean + 10 * standard deviation of the blanks. Experimental calculations of the LOD and LOQ values is detailed in Table 2.

Table 2. Mathematical definitions of Limits of Detection (LOD) and Limits of Quantification (LOQ).

  LOD LOQ
Equations for the calculations

 

Where:
k = 3
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

 

Where:
k = 10
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

The calculated values for LOD and LOQ for both nucleic acids and proteins, exemplified with dsDNA and BSA are reported in Table 3.

Table 3. Limits of Detection (LOD) and Limits of Quantification (LOQ) calculated for nucleic acids (dsDNA) and proteins (BSA) using the µDrop Plate or µDrop Duo Plates.

    µDrop Plate µDrop Duo Plate
Nucleic acid (dsDNA) LOD 2.0 µg/mL* 2.9 µg/mL*
LOQ 6.5 µg/mL * 9.8 µg/mL *
Protein (BSA) LOD 0.4 mg/ml 0.4 mg/ml
LOQ 1.5 mg/ml 1.5 mg/ml

*This concentration difference between µDrop and µDrop Duo plate is statistically meaningless because it is caused by random approx. 0.001 Abs difference in the measured Abs 260 nm value. This A260 nm difference is below the precision specification of the instrument and therefore the difference in LOD and LOQ values between these two µDrop plate types is individual random happening in this test.


Linearity ranges

The dsDNA experiment described above is also used as an example to demonstrate the linear range of the Multiskan SkyHigh and µDrop plate system in practice.

As mentioned in the detection range section, with a measurement device with fixed pathlength, like the µDrop plates, the maximum measurement range is always determined by the instrument. The lower part is determined by the precision of the blank (LOD) and the upper part by the linear range of the instrument. So, for Multiskan SkyHigh, which is specified to be linear up to 2.5 Abs, the theoretical maximum concentration is 2.5 x 50 μg/ml / 0.050= 2500 μg/ml

The absorbance values of both µDrop and µDrop Duo plates are plotted as function of the theoretical concentration of the dsDNA samples in Figure 5.

Graph of absorbance vs concentration for µDrop and µDrop Duo plates compared to theoretical concentrations of sample

Figure 5. µDrop and µDrop Duo plate absorbance A260 nm values as function of the theoretical dsDNA concentration.

This data shows that the linear range up to 2.5 Abs well applies also to Multiskan SkyHigh with the µDrop plates in dsDNA A260 nm measurements.


Conclusions

Multiskan SkyHigh combined with μDrop or μDrop Duo Plate is an ideal tool for quick and easy concentration measurements of DNA, RNA, protein samples, or spectral scanning with very low sample consumption. Multiskan SkyHigh, µDrop plates and ready-made sessions for nucleic acid and protein analysis in the UI and in the SkanIt cloud library offer a very easy to use approach to these measurements. Samples are easy to pipette onto the μDrop Plates with a single or an 8-channel pipette. The plates are easily wiped clean, making it convenient to be used in serial measurements.

Read about the µDrop and µDrop Duo plates

Key features

Fast, easy nucleic acid and protein sample measurements in low volumes

Our µDrop and µDrop Duo Plate is designed for rapid measurement of low sample volumes. You can analyze up to 16 samples simultaneously. Users can quickly and easily wipe off the samples for serial measurements. SkanIt software sessions are available for nucleic acid analysis. The μDrop Plate also contains a dedicated holder for cuvette measurements.

Simple-to-use software enables experiment setup within minutes

SkanIt software can be used to control the Multiskan SkyHigh spectrophotometer and supports optimal use of the instrument’s features with a visual workflow, easy data analysis, and exporting capabilities. SkanIt software is a multi-instrument microplate reader software that provides excellent usability and flexibility even for the most challenging assays.

Access and manage your data remotely

The Multiskan SkyHigh instrument touch screen models can connect to Thermo Fisher Connect and Microsoft OneDrive cloud-based tools to help ensure secure digital data management, permit remote access to your photometric data, and allow sharing between colleagues—all from the comfort of your computer.

Thermo Fisher Connect cloud-based capabilities enable you to:

  • Monitor the status of your instrument from your PC or Mac® computer.
  • Monitor the status of your instrument from your mobile device with the Instrument Connect app.
  • Monitor the progress of a kinetic run (e.g., bacterial growth) from your cloud account or from your mobile device with the Instrument Connect app.
  • Upload measured data automatically or manually from your instrument to your cloud account.
  • Securely store, access, share, and manage your data remotely.
  • Receive automatic notification of new instrument software updates.
  • Upgrade instrument software at your convenience.
     

Technical specifications

Wavelength selection Monochromator
Light source Xenon flash lamp
Wavelength range 200–1,000 nm with 1 nm steps
Read-out range Up to 4 absorbance units
Bandwidth <2.5 nm
Linearity at 450 nm* 0–2.5 Abs, 2%
Accuracy at 450 nm* 1.0% + 0.003 Abs (0–2.0 Abs)
2.0% (2.0–2.5 Abs)
Precision at 450 nm* SD <0.003 Abs or CV <1.0%
Plates 6-48, 96, and 384 well plates†, Thermo Scientific µDrop Plates
Cuvette Dimensions: 12.5 (W) x 12.5 (D) x 40-58 (H) mm
Beam center height: 8.5 mm
Beam window: ≥2 mm
Measurement speed (from A1 back to A1) 6 s with 96-well plate
10 s with 384-well plate
Plate shaking Linear
Spectral scanning speed 10 s from 200–1,000 nm with 1 nm steps
Incubation range From ambient + 2°C to 45°C
User interfaces Stand-alone use: 7 in. touch screen display
PC control: SkanIt software
Connections 1 USB B port for PC
1 Ethernet port
3 USB A ports for devices (USB memory device and Wi-Fi dongle)
Main input 100–240 V (50/60 Hz)
Max power consumption <110 W
Power save consumption** 7 W
Dimensions (H x W x D) 265 x 295 x 445 mm [10.4 x 11.6 x 17.5 in]
Weight 11.3 kg [24.9 lbs]
* Specifications are valid for 96-well flat bottom microplates and quartz semimicro cuvettes with a 4 mm beam window.
** Models with touch screen. †Maximum plate height with lid is 19.5 mm.

Low-volume quantification of nucleic acids and proteins with Thermo Scientific µDrop and µDrop Duo Plates

Introduction

Photometry in the UV range is a common way to quantify nucleic acids and proteins in a sample. Both nucleic acids and proteins absorb UV light. In the case of nucleic acids, the nitrogenous bases present in all nucleotides have a clearly distinguishable absorption maximum at 260 nm. On the other hand, in the case of proteins, the light absorptive properties at 280 nm are limited to few amino acids, specifically the aromatic amino acids tryptophan (Trp) and tyrosine (Tyr), and to a lesser extent cysteine groups forming disulfide bonds (Cys-Cys). Therefore, the absorption of proteins and peptides at 280 nm is proportional to the content of these amino acids.

These UV-absorptive properties make it possible to use direct photometry to quantify the concentrations of nucleic acids and proteins. Such calculations are performed using Beer-Lambert’s Law, which describes that the absorbance of a certain nucleic acid or a protein depends on the molecule’s concentration, the molecule’s absorptivity coefficient (given by the intrinsic absorptivity properties of such molecule) and the pathlength of the incident light. The use of this equation specifically for calculation of nucleic acids and proteins is shown on Table 1.

Table 1. Calculation of nucleic acids and protein concentrations using direct absorbance measurements, according to Beer-Lambert’s Law.

  Nucleic acids Proteins
Example case Double stranded DNA (dsDNA) Bovine serum albumin (BSA)
Equation for the calculations

Where:
C = concentration of dsDNA (µg/mL)
A260 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
εdsDNA = extinction coefficient of dsDNA = 0.02 (µg/mL)-1cm-1
Note: Ratio of is 50 (µg/mL) cm

Where:
C = concentration of BSA (mg/mL)
A280 = absorbance value at 260 nm of sample and blank
A320 = absorbance value at 320 nm of sample and blank
L = light pathlength (cm)
ε1% = mass extinction coefficient of BSA = 6.7 (mg/mL)-1cm-1 for 1% (10 mg/mL) BSA solution

Equation for molar extinction coefficient for ε proteins   Molar extinction coefficient ε for any protein can be easily calculated using following equation:
ε=(nW * 5500) + (nY * 1490) + (nC * 125)
Where:
W: tryptophan, Y: tyrosine, C: cysteine
n: number of each residue present in the protein
(5500, 1490, and 125: molar absorptivity at 280 nm of W, Y, and C, respectively)

The calculations of nucleic acids and proteins are performed based upon absorbance measurements at 260 nm and 280 nm, respectively, but an additional measurement at 320 nm is typically carried out with the purpose of background subtraction. Measuring at 320 nm allows excluding impurities in the sample, for example arising from the presence of magnetic beads in the sample.
 

Low-volume quantification with Thermo Scientific µDrop Plate and µDrop Duo Plates

The amount of sample available for nucleic acid and protein analysis is often low. To address that challenge, Thermo Scientific offers the µDrop and µDrop Duo Plates, which enable direct photometric measurements at a microliter scale (Figure 1). The Drop and µDrop Duo Plates consist of two separate measurement locations: the left side is intended for measurements of low-sample volumes and the right side is for measuring cuvettes. The cuvette slot is used to perform photometric measurements (including kinetic studies) with standard cuvettes that are covered with stopper and placed in a horizontal position.

The low-volume measurement area consists of two or four quartz slides: one or two pairs of the top clear quartz slide and the bottom quartz slide, which is partially Teflon-coated. The bottom slide contains a matrix array position where samples are pipetted. In the case of the µDrop Plate, there are 16 samples positions arranged in a 2 x 8 matrix, while in the case of the µDrop Duo Plate, there are 32 sample positions arranged in 2 separated arrays also in a 2 x 8 matrix (Figure 1). The pathlength of the µDrop Plate is fixed, and the nominal value is 0.05 cm. However, the pathlength can vary between 0.048 and 0.052 cm, and verified pathlength for each μDrop Plate is indicated on the quality control measurement report delivered with the μDrop Plate. Same applies to the µDrop Duo Plate (with 32 samples), but in this case, there are two separate pathlength values, one for each 2 x 8 array. These exact pathlength values should always be used for the concentration calculations, instead of the nominal value of 0.05 cm, in order to obtain more accurate results.

top view of the plates showing the quartz slides and the positions for pipetted samples

Figure 1. μDrop Plate and the μDrop Duo Plate.

Compared to a normal cuvette having a pathlength of 1 cm, the pathlength of the μDrop Plate or μDrop Duo Plate is 20 times shorter (0.05 cm). Because of this, there is roughly a 20 times difference between the concentrations that can be measured with a typical cuvette versus the μDrop and μDrop Duo Plate. This means that the detection limit of any given analyte is about 20 times higher in the μDrop and μDrop Duo Plate than with cuvettes, when measured in the same instrument. On the other hand, a shorter pathlength allows for lower sample volumes to be measured. In the case of the μDrop and μDrop Duo Plate, a sample volume down to 2 µL can be used. It is possible to measure nucleic acid and protein concentrations from a few to thousands of micrograms per microliter using these devices together with a photometer that has sufficiently high precision and a wide linear range, such as the Thermo Scientific Multiskan SkyHigh Microplate Spectrophotometer. Finally, any photometric measurement device always has a certain background absorption. Therefore, blank subtraction is necessary when photometric quantification of the sample concentrations is performed.


Materials and methods

To calculate the detection limits for nucleic acid quantification (dsDNA), 11 dilutions were made of a DNA stock solution of Herring sperm DNA (Promega, D1816) using TE-buffer, (Invitrogen 10 mM Tris-HCl, 0.1 mM EDTA), to cover concentration range between 50 and 3500 µg/mL. To calculate the detection limits for protein quantification (BSA), 9 dilutions were made of a BSA stock solution (Sigma, A7030) to distilled water to cover concentration range between 0.5 and 100 mg/ml. The blanks as well as dsDNA or BSA samples were measured by pipetting 4 µL into the measurement areas of the μDrop Plate and the µDrop Duo Plate. In case of both μDrop and µDrop duo plates, 2 blanks were used. All measurements were performed using Multiskan SkyHigh Microplate Spectrophotometer with touchscreen and cuvette.

As indicated earlier, to ensure that proper concentration calculations, the exact pathlength values of the μDrop Plate or µDrop Duo Plate need to be provided. These values were defined in Multiskan SkyHigh settings by selecting “Settings” from the Home screen and then selecting “μDrop Plates” as indicated in Figure 2. The final concentrations reported by the instrument will be calculated with these pathlength values, and the user will not need to perform any correction steps, afterwards. Ready-made assay protocols in the Multiskan SkyHigh user interface for dsDNA or BSA quantification were used to perform the assays.

User interface screenshot showing how users specify pathlengths into the software

Figure 2. Setting-up the pathlength values of the μDrop Plate and μDrop Duo Plate in the User Interface of the Multiskan SkyHigh Microplate Spectrophotometer.

When the Multiskan SkyHigh model without touch screen is used, the dsDNA concentration calculations need to be done using the Thermo Scientific SkanIt software, which controls all the Thermo Scientific microplate readers. For that purpose, in “Plate Layout”, the appropriate plate format should be selected from the dropdown menu (μDrop Plate or μDrop Duo Plate). In the case of the μDrop Duo Plate, it is possible to add two values as the pathlengths on the two separate measurement areas may differ. See an example of the performed calculations in Figure 3.

Screenshot of software showing the results of the dsDNA concentration calculations

Figure 3. Setting-up the pathlength values of the μDrop Duo Plate in the SkanIt software for calculations of the dsDNA concentrations using Multiskan SkyHigh Microplate Spectrophotometer. If the calculation is performed without 320 nm subtraction, the factor 50 is simply added to the pathlength correction step of the 260 nm measurement data.

Multiskan SkyHigh models with UI, the ready-made sessions have all the necessary calculations. The results automatically contain the concentration, purity ratios and the sample spectrum, Figure 4 below.

Calculations results screen showing the dsDNA measurements and corresponding concentration and purity values

Figure 4. An example data set of a µDrop duo plate dsDNA measurement.


Results and discussion

Detection range of the µDrop Plate and µDrop Duo Plates

The detection range is given by the lowest and highest possible concentrations that can be reliably measured with a given instrument. Therefore, these two extremes of the detection range are instrument dependent and they can be theoretically calculated from the instrument’s performance specifications. In this note, however, we focus on the detection ranges of nucleic acids and proteins, as experimentally measured using IUPAC recommendations. Often the limiting factor is the lowest end of the detection range, since unknown samples that have too low concentrations of nucleic acids or proteins (falling under the lowest part of the detection range) cannot be simply accurately estimated using photometric detection with the µDrop Plate or µDrop Duo Plates. Often, because most microplate spectrophotometers in the market have very similar detection ranges, the only solution in such cases is to change the detection technology and use instead fluorescence-based detection, which is known to be considerably more sensitive. By contrast, for samples with too high concentration values (falling outside of the detection range), a simple solution is to dilute the samples, so that they can fall within the detection range, and thus can be accurately measured.

Limits of Detection (LOD) and Limits of Quantification (LOQ) for measuring nucleic acids and proteins

The lowest part of the detection range curve indicates the assay sensitivity, which can be assessed, according to the IUPAC, with two different parameters: Limit of Detection (LOD) and Limit of Quantification (LOQ). The LOD is defined as the lowest quantity or concentration of analyte that can be separated from the background or blank values. The LOD value means that the presence of the analyte can be detected with statistical significance, but the analyte cannot be quantified as an exact value. On the other hand, the LOQ is defined as the lowest quantity or concentration of the analyte at which quantification is possible with statistical relevance. In practice, LOD is the limiting value in qualitative assays where the question “is there any analyte in my sample or not?” needs to be answered. In such cases, LOQ is the limiting value that the user can measure as the concentration of the analyte in quantitative assays. Both LOD and LOQ calculations assume that both blank samples and replicates of the analyte are normally distributed. Signals at the LOD are defined as the blank mean + 3 * standard deviation of the blank, while signals at the LOQ are defined as blank mean + 10 * standard deviation of the blanks. Experimental calculations of the LOD and LOQ values is detailed in Table 2.

Table 2. Mathematical definitions of Limits of Detection (LOD) and Limits of Quantification (LOQ).

  LOD LOQ
Equations for the calculations

 

Where:
k = 3
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

 

Where:
k = 10
SD blanks = standard deviations of instrument readings taken on assay blanks
m = slope of a graph of instrument’s blanked signal vs. concentration of the analyte on the linear range, as calculated by linear regression

The calculated values for LOD and LOQ for both nucleic acids and proteins, exemplified with dsDNA and BSA are reported in Table 3.

Table 3. Limits of Detection (LOD) and Limits of Quantification (LOQ) calculated for nucleic acids (dsDNA) and proteins (BSA) using the µDrop Plate or µDrop Duo Plates.

    µDrop Plate µDrop Duo Plate
Nucleic acid (dsDNA) LOD 2.0 µg/mL* 2.9 µg/mL*
LOQ 6.5 µg/mL * 9.8 µg/mL *
Protein (BSA) LOD 0.4 mg/ml 0.4 mg/ml
LOQ 1.5 mg/ml 1.5 mg/ml

*This concentration difference between µDrop and µDrop Duo plate is statistically meaningless because it is caused by random approx. 0.001 Abs difference in the measured Abs 260 nm value. This A260 nm difference is below the precision specification of the instrument and therefore the difference in LOD and LOQ values between these two µDrop plate types is individual random happening in this test.


Linearity ranges

The dsDNA experiment described above is also used as an example to demonstrate the linear range of the Multiskan SkyHigh and µDrop plate system in practice.

As mentioned in the detection range section, with a measurement device with fixed pathlength, like the µDrop plates, the maximum measurement range is always determined by the instrument. The lower part is determined by the precision of the blank (LOD) and the upper part by the linear range of the instrument. So, for Multiskan SkyHigh, which is specified to be linear up to 2.5 Abs, the theoretical maximum concentration is 2.5 x 50 μg/ml / 0.050= 2500 μg/ml

The absorbance values of both µDrop and µDrop Duo plates are plotted as function of the theoretical concentration of the dsDNA samples in Figure 5.

Graph of absorbance vs concentration for µDrop and µDrop Duo plates compared to theoretical concentrations of sample

Figure 5. µDrop and µDrop Duo plate absorbance A260 nm values as function of the theoretical dsDNA concentration.

This data shows that the linear range up to 2.5 Abs well applies also to Multiskan SkyHigh with the µDrop plates in dsDNA A260 nm measurements.


Conclusions

Multiskan SkyHigh combined with μDrop or μDrop Duo Plate is an ideal tool for quick and easy concentration measurements of DNA, RNA, protein samples, or spectral scanning with very low sample consumption. Multiskan SkyHigh, µDrop plates and ready-made sessions for nucleic acid and protein analysis in the UI and in the SkanIt cloud library offer a very easy to use approach to these measurements. Samples are easy to pipette onto the μDrop Plates with a single or an 8-channel pipette. The plates are easily wiped clean, making it convenient to be used in serial measurements.

Read about the µDrop and µDrop Duo plates

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