Tag Archives: biosensing

Nanopore-tal enables cells to talk to computers?

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An August 25, 2021 news item on ScienceDaily announced research that will allow more direct communication between cells and computers,

Genetically encoded reporter proteins have been a mainstay of biotechnology research, allowing scientists to track gene expression, understand intracellular processes and debug engineered genetic circuits.

But conventional reporting schemes that rely on fluorescence and other optical approaches come with practical limitations that could cast a shadow over the field’s future progress. Now, researchers at the University of Washington and Microsoft have created a “nanopore-tal” into what is happening inside these complex biological systems, allowing scientists to see reporter proteins in a whole new light.

The team introduced a new class of reporter proteins that can be directly read by a commercially available nanopore sensing device. The new system ― dubbed “Nanopore-addressable protein Tags Engineered as Reporters” or “NanoporeTERs” ― can detect multiple protein expression levels from bacterial and human cell cultures far beyond the capacity of existing techniques.

An August 12, 2021 University of Washington news release (also on EurekAlert but published August 24, 2021), which originated the news item, provides more detail (Note: Links have been removed),

“NanoporeTERs offer a new and richer lexicon for engineered cells to express themselves and shed new light on the factors they are designed to track. They can tell us a lot more about what is happening in their environment all at once,” said co-lead author Nicolas Cardozo, a doctoral student with the UW Molecular Engineering and Sciences Institute. “We’re essentially making it possible for these cells to ‘talk’ to computers about what’s happening in their surroundings at a new level of detail, scale and efficiency that will enable deeper analysis than what we could do before.”

For conventional labeling methods, researchers can track only a few optical reporter proteins, such as green fluorescent protein, simultaneously because of their overlapping spectral properties. For example, it’s difficult to distinguish between more than three different colors of fluorescent proteins at once. In contrast, NanoporeTERs were designed to carry distinct protein “barcodes” composed of strings of amino acids that, when used in combination, allow at least ten times more multiplexing possibilities. 

These synthetic proteins are secreted outside of a cell into the surrounding environment, where researchers can collect and analyze them using a commercially available nanopore array. Here, the team used the Oxford Nanopore Technologies MinION device. 

The researchers engineered the NanoporeTER proteins with charged “tails” so that they can be pulled into the nanopore sensors by an electric field. Then the team uses machine learning to classify the electrical signals for each NanoporeTER barcode in order to determine each protein’s output levels.

“This is a fundamentally new interface between cells and computers,” said senior author Jeff Nivala, a UW research assistant professor in the Paul G. Allen School of Computer Science & Engineering. “One analogy I like to make is that fluorescent protein reporters are like lighthouses, and NanoporeTERs are like messages in a bottle. 

“Lighthouses are really useful for communicating a physical location, as you can literally see where the signal is coming from, but it’s hard to pack more information into that kind of signal. A message in a bottle, on the other hand, can pack a lot of information into a very small vessel, and you can send many of them off to another location to be read. You might lose sight of the precise physical location where the messages were sent, but for many applications that’s not going to be an issue.”

As a proof of concept, the team developed a library of more than 20 distinct NanoporeTERs tags. But the potential is significantly greater, according to co-lead author Karen Zhang, now a doctoral student in the UC Berkeley-UCSF bioengineering graduate program.

“We are currently working to scale up the number of NanoporeTERs to hundreds, thousands, maybe even millions more,” said Zhang, who graduated this year from the UW with bachelor’s degrees in both biochemistry and microbiology. “The more we have, the more things we can track.

“We’re particularly excited about the potential in single-cell proteomics, but this could also be a game-changer in terms of our ability to do multiplexed biosensing to diagnose disease and even target therapeutics to specific areas inside the body. And debugging complicated genetic circuit designs would become a whole lot easier and much less time-consuming if we could measure the performance of all the components in parallel instead of by trial and error.”

These researchers have made novel use of the MinION device before, when they developed a molecular tagging system to replace conventional inventory control methods. That system relied on barcodes comprising synthetic strands of DNA that could be decoded on demand using the portable reader. 

This time, the team went a step farther.

“This is the first paper to show how a commercial nanopore sensor device can be repurposed for applications other than the DNA and RNA sequencing for which they were originally designed,” said co-author Kathryn Doroschak, a computational biologist at Adaptive Biotechnologies who completed this work as a doctoral student at the Allen School. “This is exciting as a precursor for nanopore technology becoming more accessible and ubiquitous in the future. You can already plug a nanopore device into your cell phone. I could envision someday having a choice of ‘molecular apps’ that will be relatively inexpensive and widely available outside of traditional genomics.”

Additional co-authors of the paper are Aerilynn Nguyen at Northeastern University and Zoheb Siddiqui at Amazon, both former UW undergraduate students; Nicholas Bogard at Patch Biosciences, a former UW postdoctoral research associate; Luis Ceze, an Allen School professor; and Karin Strauss, an Allen School affiliate professor and a senior principal research manager at Microsoft. This research was funded by the National Science Foundation, the National Institutes of Health and a sponsored research agreement from Oxford Nanopore Technologies. 

Here’s a link to and a citation for the paper,

Multiplexed direct detection of barcoded protein reporters on a nanopore array by Nicolas Cardozo, Karen Zhang, Kathryn Doroschak, Aerilynn Nguyen, Zoheb Siddiqui, Nicholas Bogard, Karin Strauss, Luis Ceze & Jeff Nivala. Nature Biotechnology (2021) DOI: https://doi.org/10.1038/s41587-021-01002-6 Published: 12 August 2021

This paper is behind a paywall.

Nanomushroom sensors

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Schematic illustration of cells (blue mountain-like shapes) on top of nanoscale mushroom-like structures with silicone dioxide stems and gold caps, which have the potential to detect cell proliferation in real-time. Courtesy: OIST

The nanomushroom sensors depicted in the above illustration and announced in a February 23, 2018 news item on Nanowerk probably aren’t edible but they certainly make up for that deficiency with other properties,

A small rectangle of pink glass, about the size of a postage stamp, sits on Professor Amy Shen’s desk. Despite its outwardly modest appearance, this little glass slide has the potential to revolutionize a wide range of processes, from monitoring food quality to diagnosing diseases.

The slide is made of a ‘nanoplasmonic’ material — its surface is coated in millions of gold nanostructures, each just a few billionths of a square meter in size. Plasmonic materials absorb and scatter light in interesting ways, giving them unique sensing properties. Nanoplasmonic materials have attracted the attention of biologists, chemists, physicists and material scientists, with possible uses in a diverse array of fields, such as biosensing, data storage, light generation and solar cells.

A February 23, 2018 Okinawa Institute of Science and Technology Graduate University (OIST) press release (also on EurekAlert), which originated the news item, provides more detail,

In several recent papers, Prof. Shen and colleagues at the Micro/Bio/Nanofluidics Unit at the Okinawa Institute of Science and Technology (OIST), described their creation of a new biosensing material that can be used to monitor processes in living cells.

“One of the major goals of nanoplasmonics is to search for better ways to monitor processes in living cells in real time,” says Prof. Shen. Capturing such information can reveal clues about cell behavior, but creating nanomaterials on which cells can survive for long periods of time yet don’t interfere with the cellular processes being measured is a challenge, she explains.

Counting Dividing Cells

One of the team’s new biosensors is made from a nanoplasmonic material that is able to accommodate a large number of cells on a single substrate and to monitor cell proliferation, a fundamental process involving cell growth and division, in real time. Seeing this process in action can reveal important insights into the health and functions of cells and tissues.

Researchers in OIST’s Micro/Bio/Nanofluidics Unit described the sensor in a study recently published in the journal Advanced Biosystems [citation and link follow this press release].

The most attractive feature of the material is that it allows cells to survive over long time periods. “Usually, when you put live cells on a nanomaterial, that material is toxic and it kills the cells,” says Dr. Nikhil Bhalla, a postdoctoral researcher at OIST and first author of the paper. “However, using our material, cells survived for over seven days.” The nanoplasmonic material is also highly sensitive: It can detect an increase in cells as small as 16 in 1000 cells.

The material looks just like an ordinary pieces of glass. However, the surface is coated in tiny nanoplasmonic mushroom-like structures, known as nanomushrooms, with stems of silicon dioxide and caps of gold. Together, these form a biosensor capable of detecting interactions at the molecular level.

The biosensor works by using the nanomushroom caps as optical antennae. When white light passes through the nanoplasmonic slide, the nanomushrooms absorb and scatter some of the light, changing its properties. The absorbance and scattering of light is determined by the size, shape and material of the nanomaterial and, more importantly, it is also affected by any medium in close proximity to the nanomushroom, such as cells that have been placed on the slide. By measuring how the light has changed once it emerges through the other side of the slide, the researchers can detect and monitor processes occurring on the sensor surface, such as cell division.

“Normally, you have to add labels, such as dyes or molecules, to cells, to be able to count dividing cells,” says Dr. Bhalla. “However, with our method, the nanomushrooms can sense them directly.”

Scaling Up

This work builds on a new method, developed by scientists at the Micro/Bio/Nanofluidics Unit at OIST, for fabricating nanomushroom biosensors. The technique was published in the journal ACS Applied Materials and Interfaces in December 2017.

Producing large-scale nanoplasmonic materials is challenging because it is difficult to ensure uniformity across the entire material surface. For this reason, biosensors for routine clinical examinations, such as disease testing, are still lacking.

In response to this problem, the OIST researchers developed a novel printing technique to create large-scale nanomushroom biosensors. With their method, they were able to develop a material consisting of approximately one million mushroom-like structures on a 2.5cm by 7.5cm silicon dioxide substrate.

“Our technique is like taking a stamp, covering it with ink made from biological molecules, and printing onto the nanoplasmonic slide,” says Shivani Sathish, a PhD student at OIST and co-author of the paper. The biological molecules increase the sensitivity of the material, meaning it can sense extremely low concentrations of substances, such as antibodies, and thus potentially detect diseases in their earliest stages.

“Using our method, it is possible to create a highly sensitive biosensor that can detect even single molecules,” says Dr. Bhalla, first author of the paper.

Plasmonic and nanoplasmonic sensors offer important tools for many fields, from electronics to food production to medicine. For example, in December 2017, second year Ph.D student Ainash Garifullina from the Unit developed a new plasmonic material for monitoring the quality of food products during the manufacturing process. The results were published in the journal Analytical Methods.

Prof. Shen and her unit say that, in the future, nanoplasmonic materials may even be integrated with emerging technologies, such as wireless systems in microfluidic devices, allowing users to take readings remotely and thereby minimizing the risk of contamination.

Here’s a link to and a citation for the paper,

Large-Scale Nanophotonic Structures for Long-Term Monitoring of Cell Proliferation by Nikhil Bhalla, Shivani Sathish, Abhishek Sinha, and Amy Q. Shen. Advanced Biosystems Vol. 2 Issue 2 DOI: 10.1002/adbi.201700258 Version of Record online: 19 JAN 2018

© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

This paper is behind a paywall.

Here’s a link to and a citation for the earlier paper,

Probing specific gravity in real-time with graphene oxide plasmonics by Ainash Garifullina, Nikhil Bhalla, and Amy Q. Shen. Anal. Methods 2018, 10, 290-297 DOI: 10.1039/C7AY02423A first published [online] on 06 Dec 2017

This paper is open access provided you have registered for a free account.

An easier, cheaper way to diagnose Ebola

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A Sept. 9, 2015 news item on Nanotechnology Now highlights a new technology for diagnosing the Ebola virus,

A new Ebola test that uses magnetic nanoparticles could help curb the spread of the disease in western Africa. Research published in Biosensors and Bioelectronics shows that the new test is 100 times more sensitive than the current test, and easier to use. Because of this, the new test makes it easier and cheaper to diagnose cases, enabling healthcare workers to isolate patients and prevent the spread of Ebola.

The authors of the study, from the Chinese Academy of Sciences, say their new technology could be applied to the detection of any biological molecules, making it useful to diagnose other infectious diseases, like flu, and potentially detect tumors and even contamination in wastewater.

A Sept. 9, 2015 Elsevier press release, which originated the news item, provides more detail,

The Ebola virus causes an acute illness that is deadly in half of all cases, on average. The current outbreak of Ebola, which started in March 2014, affects countries in west Africa. In the most severely affected countries, like Guinea, Liberia and Sierra Leone, resources are limited, making control of the outbreak challenging. There is no vaccine for Ebola, so detecting the virus is key to controlling the outbreak: with an accurate diagnosis, patients can be isolated and treated properly, reducing the risk of spread.

“In west Africa, resources are under pressure, so complicated, expensive tests are not very helpful,” said Professor Xiyun Yan, one of the authors of the study from the Chinese Academy of Sciences. “Our new strip test is a simple, one-step test that is cheap and easy to use, and provides a visible signal, which means people don’t need training to use it. We think it will be especially helpful in rural areas, where technical equipment and skills are not available.”

Currently there are two ways to test for the Ebola virus: using a method called polymerase chain reaction (PCR), which makes copies of the molecules for detection, and with antibody-capture enzyme-linked immunosorbent assay (ELISA), which gives a visual indication when a given molecule is in a sample. PCR is very sensitive, but is expensive and complicated, requiring special skills and technical equipment. The ELISA – or gold strip test – is cheaper but sensitivity is very low, which means it often gives the wrong results.

The new test, called the nanozyme test, uses magnetic nanoparticles, which work like enzymes to make the signal stronger, giving a clearer result you can see with the naked eye. The test can detect much smaller amounts of the virus, and is 100 times more sensitive than the gold strip test.

“People have loved the strip test for many years, but it has a major weakness: it’s not sensitive enough. We’re very excited about our new nanozyme test, as it is much more sensitive and you don’t need any specialist equipment to get a quick, accurate result,” said Dr. Yan.

Strip tests work by attaching molecules called antibodies to gold particles to look for a particular molecule in a sample. When they attach to the molecule you’re looking for, in this case a virus, they produce a signal, such as a color change. In order to find the virus, the particles need to be labelled with enzymes, which speed up detection and signalling.

With the new nanozyme test, the researchers applied magnetic nanoparticles as a nanozyme probe in place of gold nanoparticles. After labeling with an antibody that attaches to the Ebola virus, this novel probe is able to recognize and separate the virus in a sample. The nanoparticles are magnetic, so to concentrate the virus particles in a sample, all you need to do is hold the sample against a magnet; no expensive equipment is needed.

The nanozyme test is 100 times more sensitive than the gold strip test, detecting molecules called glycoproteins on the surface of the Ebola virus at concentrations as low as 1 nanogram per milliliter.

The researchers have applied for a patent for the new test, which is currently being taken to west Africa by the CDC to use in the field. The researchers are also collaborating with clinical teams to apply the technology to other diseases, and with a company that treats wastewater to see if it can help remove environmental contamination.

Here’s a link to and a citation for the paper,

Nanozyme-strip for rapid local diagnosis of Ebola by Demin Duan, Kelong Fan, Dexi Zhang, Shuguang Tan, Mifang Liang, Yang Liu, Jianlin Zhang, Panhe Zhang, Wei Liu, Xiangguo Qiu, Gary P. Kobinger, George Fu Gao, Xiyun Yan. Biosensors and Bioelectronics Volume 74, 15 December 2015, Pages 134–141 doi:10.1016/j.bios.2015.05.025

This paper appears to be open access.

Sponges made of nanoporous gold and DNA detection

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This work from the University of California at Davis seems to represent a step forward for better detection of diseases and pathogens. From a Sept. 4, 2015 news item on ScienceDaily,

Sponge-like nanoporous gold could be key to new devices to detect disease-causing agents in humans and plants, according to UC Davis researchers.

In two recent papers in Analytical Chemistry, a group from the UC Davis Department of Electrical and Computer Engineering demonstrated that they could detect nucleic acids using nanoporous gold, a novel sensor coating material, in mixtures of other biomolecules that would gum up most detectors. This method enables sensitive detection of DNA [deoxyribonucleic acid] in complex biological samples, such as serum from whole blood.

A Sept. 4, 2015 UC Davis news release on EurekAlert, which originated the news item, offers more detail,

“Nanoporous gold can be imagined as a porous metal sponge with pore sizes that are a thousand times smaller than the diameter of a human hair,” said Erkin Şeker, assistant professor of electrical and computer engineering at UC Davis and the senior author on the papers. “What happens is the debris in biological samples, such as proteins, is too large to go through those pores, but the fiber-like nucleic acids that we want to detect can actually fit through them. It’s almost like a natural sieve.”

Rapid and sensitive detection of nucleic acids plays a crucial role in early identification of pathogenic microbes and disease biomarkers. Current sensor approaches usually require nucleic acid purification that relies on multiple steps and specialized laboratory equipment, which limit the sensors’ use in the field. The researchers’ method reduces the need for purification.

“So now we hope to have largely eliminated the need for extensive sample clean-up, which makes the process conducive to use in the field,” Şeker said.

The result is a faster and more efficient process that can be applied in many settings.

The researchers hope the technology can be translated into the development of miniature point-of-care diagnostic platforms for agricultural and clinical applications.

“The applications of the sensor are quite broad ranging from detection of plant pathogens to disease biomarkers,” said Şeker.

For example, in agriculture, scientists could detect whether a certain pathogen exists on a plant without seeing any symptoms. And in sepsis cases in humans, doctors might determine bacterial contamination much more quickly than at present, preventing any unnecessary treatments.

Here are links to and citations for two recent published papers about this work,

Effect of Nanoporous Gold Thin Film Morphology on Electrochemical DNA Sensing by Pallavi Daggumati, Zimple Matharu, and Erkin Şeker. Anal. Chem., 2015, 87 (16), pp 8149–8156 DOI: 10.1021/acs.analchem.5b00846 Publication Date (Web): April 30, 2015

Copyright © 2015 American Chemical Society

Biofouling-Resilient Nanoporous Gold Electrodes for DNA Sensing by Pallavi Daggumati, Zimple Matharu, Ling Wang, and Erkin Şeker. Anal. Chem., 2015, 87 (17), pp 8618–8622 DOI: 10.1021/acs.analchem.5b02969 Publication Date (Web): August 14, 2015

Copyright © 2015 American Chemical Society

These papers are behind a paywall.

Tattoos that detect glucose levels

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Temporary tattoos with a biomedical function are a popular topic and one of the latest detects glucose levels without subjecting a person with diabetes to pin pricks. From a Jan. 14, 2015 news item on ScienceDaily,

Scientists have developed the first ultra-thin, flexible device that sticks to skin like a rub-on tattoo and can detect a person’s glucose levels. The sensor, reported in a proof-of-concept study in the ACS [American Chemical Society] journal Analytical Chemistry, has the potential to eliminate finger-pricking for many people with diabetes.

A Jan. 14, 2015 ACS news release on EurekAlert, which originated the news item, describes the current approaches to testing glucose and the new painless technique,

Joseph Wang and colleagues in San Diego note that diabetes affects hundreds of millions of people worldwide. Many of these patients are instructed to monitor closely their blood glucose levels to manage the disease. But the standard way of checking glucose requires a prick to the finger to draw blood for testing. The pain associated with this technique can discourage people from keeping tabs on their glucose regularly. A glucose sensing wristband had been introduced to patients, but it caused skin irritation and was discontinued. Wang’s team wanted to find a better approach.

The researchers made a wearable, non-irritating platform that can detect glucose in the fluid just under the skin based on integrating glucose extraction and electrochemical biosensing. Preliminary testing on seven healthy volunteers showed it was able to accurately determine glucose levels. The researchers conclude that the device could potentially be used for diabetes management and for other conditions such as kidney disease.

There is a Jan. 14, 2015 University of California at San Diego news release (also on EurekAlert) describing the work in more detail,

Nanoengineers at the University of California, San Diego have tested a temporary tattoo that both extracts and measures the level of glucose in the fluid in between skin cells. …

The sensor was developed and tested by graduate student Amay Bandodkar and colleagues in Professor Joseph Wang’s laboratory at the NanoEngineering Department and the Center for Wearable Sensors at the Jacobs School of Engineering at UC San Diego. Bandodkar said this “proof-of-concept” tattoo could pave the way for the Center to explore other uses of the device, such as detecting other important metabolites in the body or delivering medicines through the skin.

At the moment, the tattoo doesn’t provide the kind of numerical readout that a patient would need to monitor his or her own glucose. But this type of readout is being developed by electrical and computer engineering researchers in the Center for Wearable Sensors. “The readout instrument will also eventually have Bluetooth capabilities to send this information directly to the patient’s doctor in real-time or store data in the cloud,” said Bandodkar.

The research team is also working on ways to make the tattoo last longer while keeping its overall cost down, he noted. “Presently the tattoo sensor can easily survive for a day. These are extremely inexpensive—a few cents—and hence can be replaced without much financial burden on the patient.”

The Center “envisions using these glucose tattoo sensors to continuously monitor glucose levels of large populations as a function of their dietary habits,” Bandodkar said. Data from this wider population could help researchers learn more about the causes and potential prevention of diabetes, which affects hundreds of millions of people and is one of the leading causes of death and disability worldwide.

People with diabetes often must test their glucose levels multiple times per day, using devices that use a tiny needle to extract a small blood sample from a fingertip. Patients who avoid this testing because they find it unpleasant or difficult to perform are at a higher risk for poor health, so researchers have been searching for less invasive ways to monitor glucose.

In their report in the journal Analytical Chemistry, Wang and his co-workers describe their flexible device, which consists of carefully patterned electrodes printed on temporary tattoo paper. A very mild electrical current applied to the skin for 10 minutes forces sodium ions in the fluid between skin cells to migrate toward the tattoo’s electrodes. These ions carry glucose molecules that are also found in the fluid. A sensor built into the tattoo then measures the strength of the electrical charge produced by the glucose to determine a person’s overall glucose levels.

“The concentration of glucose extracted by the non-invasive tattoo device is almost hundred times lower than the corresponding level in the human blood,” Bandodkar explained. “Thus we had to develop a highly sensitive glucose sensor that could detect such low levels of glucose with high selectivity.”

A similar device called GlucoWatch from Cygnus Inc. was marketed in 2002, but the device was discontinued because it caused skin irritation, the UC San Diego researchers note. Their proof-of-concept tattoo sensor avoids this irritation by using a lower electrical current to extract the glucose.

Wang and colleagues applied the tattoo to seven men and women between the ages of 20 and 40 with no history of diabetes. None of the volunteers reported feeling discomfort during the tattoo test, and only a few people reported feeling a mild tingling in the first 10 seconds of the test.

To test how well the tattoo picked up the spike in glucose levels after a meal, the volunteers ate a carb-rich meal of a sandwich and soda in the lab. The device performed just as well at detecting this glucose spike as a traditional finger-stick monitor.

The researchers say the device could be used to measure other important chemicals such as lactate, a metabolite analyzed in athletes to monitor their fitness. The tattoo might also someday be used to test how well a medication is working by monitoring certain protein products in the intercellular fluid, or to detect alcohol or illegal drug consumption.

This reminds me a little of the Google moonshot project concerning health diagnostics. Announced in Oct. 2014, that project involved swallowing a pill containing nanoparticles that would circulate through your body monitoring your health and recongregating at your wrist so a band worn there could display your health status (Oct. 30, 2014 article by Signe Brewster for GigaOm). Experts welcomed the funding while warning the expectations seemed unrealistic given the current state of research and technology. This temporary tattoo seems much better grounded in terms of the technology used and achievable results.

Here’s a link to and a citation for the paper,

Tattoo-Based Noninvasive Glucose Monitoring: A Proof-of-Concept Study by Amay J. Bandodkar, Wenzhao Jia, Ceren Yardımcı, Xuan Wang, Julian Ramirez, and Joseph Wang. Anal. Chem., 2015, 87 (1), pp 394–398 DOI: 10.1021/ac504300n Publication Date (Web): December 12, 2014

Copyright © 2014 American Chemical Society

This appears to be an open access paper.

My latest posting posting on medical tattoos (prior to this) is an Aug. 13, 2014 post about a wearable biobattery.