Oligo Modifications
Support and Educational Content

Aptamer-based electrochemical biosensors using methylene blue as a redox reporter

Methylene blue quick facts

Availability: DNA and RNA

Location: 5′, internal, and 3′

Scales: 100 nmol to large scale

Purification: HPLC required

Ordering format: /5MeBlN/ at 5′ end; email noncat@idtdna.com for internal or 3′ end requests

The proprietary synthesis platform developed and optimized at IDT not only enables quick delivery of high quality oligos, but also allows efficient incorporation of a broad array of chemical modifications into an oligo sequence. The availability of diverse modifications further empowers a myriad of experimental applications. Here we summarize historical uses of methylene blue, and highlight some of its potential applications, including use as a redox indicator.

The original synthetic “drug”

First prepared in 1876 as a chemical compound, methylene blue has been known as the “first fully synthetic drug used in medicine” [1]. In the early days, its utility was widely acknowledged as a therapeutic treatment for methemoglobinemia, an often-inherited blood disorder, as well as for malaria infection [2,3]. In addition, it has found numerous applications in pathology and cytology. As a stain, methylene blue is internalized by dead but not living cells. This property led to its use in a simple colorimetric method developed in the early 1980s to assess cell viability [4]. Methylene blue is also broadly used as a redox indicator in analytical chemistry. It produces a blue solution when dissolved in an oxidizing environment and will turn colorless when exposed to a reducing agent.

A highly sensitive redox reporter

Over the past decade methylene blue has demonstrated utility in development of aptamer-based, electrochemical biosensors for both diagnostics and basic research applications. Aptamers are synthetic oligonucleotides that bind to target molecules with high affinity and specificity through their 3-dimensional structures [5]. (For a more detailed explanation, see the sidebar, Definition: Aptamers, in the article, Planning to work with aptamers?) When used as biosensors, aptamers are frequently modified at one terminus with a redox reporter, such as methylene blue. The other terminus of the aptamer is immobilized on an electrode through other modifications, such as thiol groups (Figure 1). Upon interaction with their targets, the electrode-bound aptamers undergo conformational changes that affect electron transfer efficiency between the reporter moiety and interrogating electrode (Figure 2) [6]. In one pioneering study published in 2007, researchers immobilized the methylene blue–modified, binding-responsive oligo onto a gold surface via a thiol linkage, and then challenged the sensors with complicated biological samples. By monitoring the Faradic current produced by the interaction between the redox molecule and electrode surface, the scientists successfully signaled the target at picomolar levels [7].

an aptamer-based biosensor terminally labeled with methylene blue and thiol modifier

Figure 1. An aptamer-based biosensor terminally labeled with methylene blue and thiol modifier. MB = methylene blue.

Continuous, real-time, reusable target recognition

The above study is just one example of the novel electrochemical detection schemes that employ a methylene blue–modified aptamer as a recognition element. Thanks to the ever-improving aptamer selection strategies, it is possible to develop aptamers for virtually any sophisticated target molecule. The reversible electrochemical property of methylene blue provides the advantage of reagent-free and reusable target recognition. Without the need to wash or add exogenous reagents, the biosensors using methylene blue can function continuously and in real-time. These attributes make this class of biosensors appealing in a broad range of areas, such as point-of-care clinical diagnostics [8,9].

A methylene blue-labeled electrochemical biosensor

Figure 2. A methylene blue-labeled electrochemical biosensor. Conformational changes occurring in the oligo upon interaction with the target molecule bring the methylene blue moiety in close proximity to the electrode surface, leading to initiation of an electrical signal. MB = methylene blue.

How to order

IDT attaches methylene blue to an oligo post-synthetically. It is attached through an amino modifier, incorporated during the synthesis process, using NHS ester chemistry. Methylene blue is offered as an “off-catalog” modification when it is desired at the 5′ end of an oligo, and can be ordered directly through the IDT website by using the proper modification code, /5MeBlN/. To order an internal or 3′ methylene blue modification, please email noncat@idtdna.com with your account information, and request scale and sequence designs.


  1. Howland RH. (2016) Methylene blue: The long and winding road from stain to brain: Part 1. J Psychosoc Nurs Ment Health Serv, 54(9):21–24.
  2. Matisoff AJ, Panni MK. (2006) Methylene blue treatment for methemoglobinemia and subsequent dramatic bispectral index reduction. Anesthesiology, 105(1):228.
  3. Zoungrana A, Coulibaly B, et al. (2008) Safety and efficacy of methylene blue combined with artesunate or amodiaquine for uncomplicated falciparum malaria: a randomized controlled trial from Burkina Faso. PLoS One, 3(2):e1630.
  4. Riss TL, Moravec RA, et al. (2004) Cell Viability Assays. In: Sittampalam GS, Coussens NP, et al. (editors) Assay Guidance Manual [Internet] Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences.
  5. Sun H, Zhu X, et al. (2014) Oligonucleotide aptamers: new tools for targeted cancer therapy. Mol Ther Nucleic Acids, 3:e182.
  6. Xiao Y, Lai RY, Plaxco KW. (2007) Preparation of electrode-immobilized, redox-modified oligonucleotides for electrochemical DNA and aptamer-based sensing. Nat Protoc, 2(11):2875–2880.
  7. Schoukroun-Barnes LR, Macazo FC, et al. (2016) Reagentless, structure-switching, electrochemical aptamer-based sensors. Annu Rev Anal Chem (Palo Alto Calif), 9(1):163–181.
  8. Ferguson BS, Hoggarth DA, et al. (2013) Real-time, aptamer-based tracking of circulating therapeutic agents in living animals. Sci Transl Med, 5(213):213ra165.
  9. Liu Y, Tuleouva N, et al. (2010) Aptamer-based electrochemical biosensor for interferon gamma detection. Anal Chem, 82(19):8131–8136.

Product focus—oligos and modifications

Custom oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides, and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

Learn more or order now.

Oligo modifications

Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification request. Just send your inquiry to noncat@idtdna.com.

Additional reading

Planning to work with aptamers?—We are often asked whether IDT manufactures aptamers. The answer is, yes! IDT does synthesize aptamers and aptamer libraries, and there are already 100s of published research papers describing the successful use of such sequences manufactured by IDT. Learn about aptamers, SELEX, and how IDT can assist you with reagents for your aptamer applications.

Oligo modification—post-synthesis conjugation explained—Did you know IDT can add modifications to your oligos post-synthesis using NHS Ester chemistry? We can also add modifications through azide and alkyne groups post-synthesis, using click chemistry. Read about how these reactions are done, and get answers to common questions regarding post-synthesis conjugation.

When dT is required for modification attachment—Modification Highlight: Certain modifications require a dT base in the oligonucleotide sequence in order to be added. Learn which modifications these are, and how to add them to your oligo sequence.

Oligonucleotide modifications that block nuclease degradation—Modification Highlight: Are you working with your oligos in cells culture or in vivo? Find out which modifications can be added to an oligo to limit nuclease degradation.

Review other DECODED Online newsletter articles on oligo handling and analysis, and oligo modifications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Brian Wang, PhD, is the Genomic Tools Market Development Manager at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

IDT web tools for oligo properties

Free, online tools for oligo design, secondary structure, dilution, and resuspension.

Try them now ≫

Related Articles

DNA Oligonucleotide Resuspension and Storage

Guidelines and recommendations for how to resuspend and store newly synthesized oligonucleotides.

Read more ≫

Calculation Tips for Resuspending and Diluting Nucleic Acids

Easy guidelines for making a 100 µM solution; calculating nmoles, µg, copy number, and concentration; and determining concentration equivalencies.

Read more ≫

Understanding Melting Temperature

Advice on considerations for better oligo design: oligo concentration, salt, and SNPs.

Read more ≫

Oligonucleotide Modifications: Choosing the Right Mod for Your Needs

Guidelines on selecting specific oligonucleotide modifications and how they can help you in your research.

Read more ≫

Which Biotin Modification to Use?

Applications of each of the different biotins available from IDT.

Read more ≫