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March 26, 2020 - Do you measure your backgrounds?

In many analytical sciences, quantifying the background signal (or blank) before measuring the sample of interest is an important part of experimental design, and a critical requirement for ensuring meaningful results. A positive test signal must be compared to the background signal level in order to assess its significance and to exclude the contribution of the background from the interpretation of results. For example, in an enzyme-linked immunosorbent assay (ELISA) it's important to measure the signal from a negative control reaction that contains no target antigen, to obtain the baseline signal for other reactions.

Not everyone considers the importance of background measurements in particle analysis, though the same fundamental principles hold true. A common scenario where this is relevant is when the sample of interest must be diluted before measurement — in this case the particle content of the diluent itself must be measured and accounted for in subsequent analysis. The measured background level can also inform the measurement protocol: For example, if the sample of interest is at a concentration of 1×109 particles per mL and the background in the diluent contains 1×108 particles per mL in the same size range, then it's clear the sample cannot be diluted more than ten-fold before the measurements are no longer meaningful. These experimental bounds can only be obtained through careful measurements.

Control experiments like these are not often considered in particle analysis because of the limitations of commonly-used technology. Most techniques, including optical ones like Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS), are not additive. For these techniques, measurement of the combined sample A + B is not equal to the sum of the measurement of sample A and the measurement of sample B. Most researchers understand why Dynamic Light Scattering is not additive — it's because DLS only reports a skewed average of all the particles in the sample. More surprisingly though, since it's billed as a single-particle measurement technique, Nanoparticle Tracking Analysis isn't additive either. We and others have shown this pretty clearly, and you can easily test for yourself the next time you're in the lab (or maybe even working from home if you're so equipped!).

Spectradyne's nCS1 measures particles one-by-one, each particle truly independent from the others. As a result, Microfluidic Resistive Pulse Sensing (MRPS) is a true additive measurement technique, and thus holds enormous power for quantifying particles in complex media such as plasma, urine, or cell culture medium. Shown on the right is a case in point: a nanoparticle therapeutic needed to be quantified directly in plasma; subtracting the plasma background from the total sample yielded a direct and accurate measurement of the therapeutic particles themselves. Direct measurements in such complex media are fast and easy with MRPS — read about a pilot study we performed with Pfizer Inc., here.

Food for thought while working from home during COVID-19 isolation. Take care!

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A 96-well ELISA plate. An antibody linked to an enzyme will recognize a target protein in each well. When a colorimetric substrate is added to the sample, an enzymatic reaction causes a color change. Photo credit: Shenzhen Boomingshing Medical Device Co., LTD


This plot shows differential measurements made using Spectradyne's nCS1TM of blood plasma with and without a nanoparticle therapeutic.

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